Abstract:Abstract:A cell of gram-negative bacteria is surrounded by two layers of membrane，the inner membrane and the outer membrane．Proteins are the major composition of outer membrane．Many outer membrane proteins carry a trans-membrane β-barrel structure that formed by multiple anti-parallel β-strands connected with hydrogen bonds．These proteins can act as porins，transporters，enzymes，receptors，virulence factors and structural proteins. Therefore，their correct folding and membrane integration are important for the survival of gram-negative bacteria． Most β-barrel outer membrane proteins could be easily expressed recombinantly and refolded in vitro under certain conditions． The in vitro folding processes could be monitored and investigated through many ways，which makes outer membrane proteins become a model system to study the effects of abiotic and biological factors on the folding of membrane proteins． In this article，the research progress on the in vitro refolding of outer membrane proteins are reviewed from the aspects of refolding methods，the factors that affect folding processes and experimental methods． Finally，the research prospects in this field are discussed.

Abstract:Abstract:Salmonella enterica serotype 1，4，［5］，12: i:- ( Salmonella 1，4，［5］，12: i:-)，an emerging serotype antigenically related to Salmonella Typhimurium (1，4，［5］，12:i:1，2) but lacking the second phase flagellar antigen，has been frequently detected in many countries over the last 10 years． Nowadays it seems to be one of the major serotypes responsible for human salmonellosis cases worldwide． In addition，multidrug resistance is quite common in Salmonella 1，4，［5］，12:i:-，the two major clones (labelled as Spanish and European clones) show multidrug resistance to four or more unrelated classes of antimicrobials mediated by plasmids or chromosome． Some resistance determinants including blaTEM，blaCTX-M-1，aac(3) -IV，aadA2，cmlA1，sul1，sul2，dfrA12，strA-strB，tet (A) and tet (B) have been found in these multidrug resistance strains． The genomic characterization of 1，4，［5］，12:i:-isolates suggests that this serovar is likely to gather several clones or strains that have independently emerged from S． Typhimurium，and have changed through multiple independent events involving different clonal groups．In later study，emphasis should be paid on development of rapid and precise detection methods and study of pathogenic and resistance mechanisms of Salmonella 1，4，［5］，12:i:-．

Abstract:Abstract:［Objective］The purpose of this study is to characterize the community composition and phylogenetic analysis of cyanobacteria from supraglacial cryoconite of the Glacier No.1 in the Tianshan Mountains，China．［Methods］ We amplified 16S rＲNA genes from the extracted cryoconite DNA by PCＲ with 2 pairs of cyanobacteria-specific primers．Amplificon was used to construct 16S rＲNA genes clone library． The estimation of species richness，diversity indices，and rarefaction curve of the 16S rＲNA genes library were determined based on representative phylotypes (OTUs). ［Results］ Analysis of 16S rＲNA gene sequences allowed grouping of 101 clones into 12 phylotypes (OTUs) using a cut-off of 97% identity．The phylogenetic analysis revealed that most of sequences affiliated to the order Oscillatoriales and Chroococcales except that three were unclassified． The clone library was dominated by representatives of the order Oscillatoriales (81% of the total clones)，and the most abundant organisms within this order were in the genus Phormidium (68 clones) including clones grouping into four phylotypes． The only clone of Chroococcales was closely related to the genus Chamaesiphon with 97% similarity． In addition，comparison of soil chemical properties between different habitats indicated that supraglacial cryoconite supported significantly higher the content of available phosphorus and potassium，nitrate nitrogen and organic matter compared with the forefield of the Glacier No.1.［Conclusion］The diversity index of cyanobacteria were relatively high in supraglacial cryoconite of the Glacier No.1 in the Tianshan Mountains． The community structure was dominated by members of the genus Phormidium．This study may enrich our knowledge on biogeochemical processes and ecological distribution of cyanobacterial populations in glacial ecosystem．

Abstract:Abstract:［Objective］This study was aimed to obtain a mitogen-activated protein kinase (MAPK) gene namely FoHog1 from Fusarium oxysporum f． sp．cubense and to verify its function．［Methods］We amplified FoHog1 gene by PCR and RT-PCR methods and analyzed it through bioinformatics method．PEG-mediated protoplast transformation was used to create the deletion mutants of FoHog1 gene．We analyzed different biological characteristics between knock-out strain and wild-type strain.［Results］FoHog1 gene encoding a putative protein of 357 amino acids and its genetic relationship with different Fusarium’s protein．Compared with the wild-type strain，FoHog1 deletion mutants have loose hyphae colony， less spores production，lower dry weight of hyphae and more sensitive to temperature，pH and osmotic stress．FoHog1 deletion mutants also have reduced colonization ability compared with the wild-type strain．［Conclusion］FoHog1 gene participated in mycelial growth，sporulation，catabolism of sodium acetate and ammonium chloride，osmotic stress response and pathogenic process with Fusarium oxysporum f. sp. cubense Ｒace 4.

Abstract:Abstract:［Objective］Reactive oxygen species are natural products of metabolism in aerobic organisms，which lead to oxidative damage，such as DNA mutation，protein inactivation and drug resistance．MSMEG_3312 was predicted as a hemerythrin-like protein，which can carry oxygen and reversibly bind to oxygen，thus it might play important roles in the process of oxygen metabolism．In this study，we explored the role of MSMEG_3312 in drug resistance.［Methods］On the basis of bioinformatics，we identified the conserved sequence of HHE domain in MSMEG_3312 and it was predicted to have typical α-helix at secondary structure．To explore potential functions of MSMEG_3312，we constructed the msmeg_3312 knockout strain and compare the susceptibility to various drugs to its parent strain，mc2155．In addition，we also measured the promoter response when treatment of erythromycin．［Results］Genetic results showed that MSMEG_3312 is not necessary for M. smegmatis growth at 7H9 rich medium．The msmeg _ 3312 knockout strain showed increased erythromycin resistance． Moreover，the drug resistance is only limited to erythromycin which its mechanism of action is by binding to the 50S subunit of the bacteria ribosomal complex and then inhibit protein synthesis．However，there were no different MICs of other antibiotics，targets for protein synthesis inhibition，but not 50S subunit，such as tetracyclines，aminoglycosides and chloramphenicol．Moreover，we also showed that the promoter of msmeg_3312 responses to erythromycin． ［Conclusions］Hemerythin-like protein MSMEG_3312 is involved in erythromycin resistance.

Abstract:Abstract:［Objective］To elucidate the chemical structures of the main antibacterial compound and red pigment produced by the co-culture of marine-derived fungus Aspergillus sp．SCSGAF 0076 and bacterium Bacillus sp． MNMCCE 001．［Methods］The monoculture of strain SCSGAF 0076 and co-culture of strains SCSGAF 0076 and MNMCCE 001 were done on amylum solid medium for three days，then the crude extracts of the cultures were obtained，and subsequently，the chemical profiles of the extracts of monoculture and co-culture were analyzed by HPLC． Using antibacterial bioassayguided fractionation，we isolated the crude extract of the co-culture by silica gel column chromatogram，Sephadex LH-20，and semi-preparative HPLC to obtain the main antibacterial compound and red pigment．The compounds’structures were determined by spectroscopic analysis．［Results］ We found that the main secondary metabolites produced by the monoculture of strain SCSGAF 0076 and co-culture of strains SCSGAF 0076 and MNMCCE 001 were almost the same，however，the contents of the main antibacterial compound and red pigment were obviously different．Totally，four compounds including the antibacterial compound penicillic acid，5 (6) -dihydropenicillic acid，9-chloro-8-hydroxy-8，9- deoxyasperlactone and red pigment viopurpurin were isolated from the crude extract of the co-culture medium．［Conclusion］ The main antibacterial compound of the co-culture of strains SCSGAF 0076 and MNMCCE 001was penicillic acid，the main red pigment was viopurpurin，and the yields of the two compounds were proved by the coculture．

Abstract:Abstract:［Objective］To clone calcium-dependent protein kinase gene (camk) from Puccinia striiformis f. sp. tritici (Pst) and analyze its function.［Methods］The cDNA full-length of Pscamk was isolated by using reverse transcriptional-PCR (RT-PCR)，and gene expression profile at different morphological stages was analyzed via quantitative real-time -PCR(qRT-PCR)．Pst urediospores were treated with CaMK suppressor KN-93 and germination rate was investigated．［Results］A gene cDNA full-length with 1 620 bp was obtained and designated as Pscamk．qRT-PCR analysis showed Pscamk expression was highly induced in the early stages of Pst infection and reached the maximum at 6 h post inoculation (hpi) as 20. 74-fold as that in the control (0 hpi)．With increasing of the concentration of CaMK suppressor KN-93，germination rate of Pst urediospores was gradually decreased． The germination rate was reduced to 8.02%，only 12% of the control，under 1.4 μmol/L KN-93 treatment at 10 h after incubation at 9 ℃．［Conclusion］Pscamk might play a role in germination and germ tube elongation of Pst urediospores．This study provides a basis for exploring pathogenesis of calcium signaling pathway during Pst infection．

Abstract:Abstract:［Objective］AUＲ1 encoding inositol phosphorylceramide (IPC) synthase is the key enzyme for the sphingolipid metabolism in fungi. In this study，we explored the mechanism of AUR1 intron on the regulation of AUR1 gene expression at transcriptional and translational levels in Botrytis cinerea，as well as the influence of AUR1 intron on the pathogenicity．［Methods］AUR1 mRNA expression of wild-type B．cinerea (BcAUR1 ) and the mutant with deletion of 115 bp intron (BcAUR1a) was detected by Real-time quantitative PCR．The activity of IPC synthase from BcAUR1 and BcAUR1a was measured through high-efficiency liquid fluorescent chromatogram．In addition，H2O2 concentration and activities of superoxide dismutase (SOD)，peroxidase (POD) and catalase (CAT) per unit fungus were determined by horseradish peroxidase，pyrogallol oxidation，guaiacol and ultraviolet spectrophotometric，respectively.［Results］IPC synthase had no amino acid mutation in mutant BcAUR1a．The expression of AUR1 gene at mRNA level and the activity of IPC synthase in BcAUR1a increased by 50.2% and 14.16% compared to those in BcAUR1．The secretion of H2O2，SOD，POD and CAT in BcAUR1 was significantly stimulated by Aureobasidin A (AbA) treatment，in contrast，no significant influence was detected upon the secretion of these substances in BcAUR1a via AbA treatment.［Conclusion］The expression of AUR1 in BcAUＲ1a is significantly up-regulated at transcriptional and translational levels．AbA treatment can significantly enhance the pathogenicity of BcAUR1，but has a minor influence on the BcAUR1a． BcAUR1a is AbA-resistant．The results suggest that AUR1 gene intron regulate the expression of AUR1 as a transcriptional repressor．

Abstract:Abstract:［Objective］ This study was aimed to investigate the abundance and community shift of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in air-dried forest soils in response to water addition，to explore the applicability of air-dried soil for microbial ecology study，and to elucidate whether AOA within the marine group 1. 1a dominate ammonia oxidizers communities in the acidic forest soils in China． ［Methods］Soil samples were collected from 10 forest sites of the China Ecosystem Ｒesearch Network (CERN) and kept under air-drying conditions in 2010． In 2013 the air-dried soil samples were adjusted to 60% of soil maximum water holding capacity for a 28-day incubation at 28 ℃ in darkness． DGGE fingerprinting，clone library construction，pyrosequencing and quantitative PCR of amoA genes were performed to assess community change of ammonia oxidizers in air-dried and re-wetted soils．［Results］After incubation for 28 days，the abundance of bacteria and archaea increased significantly，up to 3，230 and 568 times，respectively．AOA increased significantly in 8 samples，and AOB increased significantly in 5 of 10 samples． However，pyrosequencing of amoA genes reveals insignificant changes in composition of AOA and AOB communities． Phylogenetic analysis of amoA genes indicates that archaeal ammonia oxidizers were predominated by AOA within the soil group 1. 1b lineage，while the Nitrosospira-like AOB dominate bacteria ammonia oxidizer communities．There was a significantly positive correlation between AOA/AOB ratio and total nitrogen (r2=0.54，P＜0.05)，implying that soil ammonia oxidation might be dominated by AOA in association with ammonium released from soil mineralization．［Conclusion］ Phylogenetic analysis suggest that AOA members within the soil group 1. 1b lineage were not restricted to non-acidic soils as previously thought．The abundance rather than composition of AOA and AOB changed in response to water addition． This indicates that air-dried soil could be of help for microbial biogeography study.

Abstract:Abstract:［Objective］ Isolation of specific bacteria from human intestinal microflora to convert naringin to naringenin efficiently． ［Methods］Fresh human feces from healthy individual was cultured in an anaerobic chamber for 24 h before being diluted and spread on agar medium．We cultured and incubated each single colony with the substrate naringin．The biotransformation of naringin by each single colony was detected by high-performance liquid chromatography (HPLC). We identified the isolated bacteria based on the analyses of 16S rDNA sequence and biochemical traits．We also studied the bioconversion kinetics of the bacteria．［Results］Four bacterial strains，named AUH-JLD3，AUH-JLD7，AUH-JLD104 and AUH-JLD109，capable of biotransforming naringin to naringenin，were isolated and identified as Blautia sp．AUHJLD3，Enterococcus sp． AUH-JLD7，Bacteroides sp．AUH-JLD104 and Streptococcus pasteurianus subsp．AUH-JLD109 respectively based on the 16S rDNA sequence analysis，bacterial cell morphology，and biochemical traits． Study on biotransforming kinetics showed that all the four isolated bacterial strains were able to convert naringin (0.2 mmol/L) to naringenin within 12 h．The maximal concentration of the substrate naringin that strain AUH-JLD3，strain AUH-JLD7，strain AUH-JLD104 and strain AUH-JLD109 could biotransform efficiently were 0.2 mmol/L (the average biotransforming rate was 66.67%)，0.8 mmol/L (the average biotransforming rate was 86.49%)，0.2 mmol/L (the average biotransforming rate was 73.68%) and 1.6 mmol/L (the average biotransforming rate was 93.20%)，respectively.［Conclusion］ The four bacterial strains were capable of biotransforming naringin to naringenin， among which Streptococcus pasteurianus subsp．AUH-JLD109 has the highest naringin biotransforming capacity.

Abstract:Abstract:［Objective］ To isolate phosphate-solubilizing microorganisms from farmland，and to provide P-solubilizing microbial resource for bio-fertilizer production．［Methods］ Phosphate-solubilizing fungus was identified using morphological and cultural characteristics and ITS rDNA sequence analysis． The phosphate-solubilizing capacity of strain P83 was measured by Petri dishes，broth medium and soil pot experiment．The effect of strain P83 on plant growth was studied in field trials．［Results］ Strain P83 was identified as Penicillium decumbens with a strong ability to dissolve insoluble phosphates．P83 dissolved 42.68% Ca3 (PO4) 2 (5g/L) and the concentration of available phosphorus was 956 mg/L during a 10-d shaking incubation．The concentration of available phosphorus dissolved from Yonghe rock phosphate by P83 was152.8mg/L after 10d shaking incubation at 28℃ and a speed of 180r/min．P．decumbens P83 had a significant growth promotion effect on corn in Chao soil under three phosphates such as Ca3 (PO4) 2，Zn3 (PO4) 2 and rock phosphate．Compared with the control，inoculation with P83 increased the fresh weight of corn biomass by 9.5%－89.2% and dry weight of corn biomass by 35%－231%，and soil available phosphorus content increased 2.1mg/kg－40.5mg/kg．Field trials show that P．decumbens P83 had a greater effect on enhancement of corn grain yield，the yield was average 9.2t/hm2 and 35.3% higher than the control．［Conclusion］One new phosphate-solubilizing strain P83 was obtained and identified as P.decumbens．It solubilized insoluble phosphates in petri dishes，broth medium and pot experiments．P．decumbens P83 could increase corn yield significantly in field trials．P．decumbens P83 strain has the potential for biofertilizer production in the future．

Abstract:Abstract:［Objective］ The fungi diversity in the guts of five sub-adult giant pandas was analyzed.［Method］ We analyzed the fungal internal transcribed spacer sequences (ITS) using restriction fragment length polymorphism (RFLP)．ITS regions were amplified with fungal universal primers to construct ITS clone libraries．The fingerprints were analyzed by restriction fragment length polymorphism using the Hha I and Hae III enzymes．The cloned PCＲ products were analyzed by sequencing and diversities were demonstrated by phylogenetic tree.［Results］The gut fungi of 5 sub-adult giant pandas were mainly composed of Ascomycota (average of 46.24%)，Basidiomycota (average of 15.79%)，unclassified (average of 29.14%)，uncultured fungus (average of 8.83%)．Ascomycota was mainly composed of Saccharomycetes (average of 63.74%) and Dothideomycetes (average of 35.91%); Basidiomycota was mainly composed of Tremellomycetes (average of 65.80%) and Microbotryomycetes (average of 33.15%)．Four classes were mainly composed of Candida and Debaryomyces; Pleosporales and Myriangium; Cystofilobasidium and Trichosporon; Leucosporidium，and Leucosporidiella，whereas the proportions were different for each sample．［Conclusion］ Fungal flora existing in the intestines of sub-adult giant pandas expand our knowledge on the structure of the giant panda gut microbes and also help us to further study whether fungal flora can help giant pandas digest high-fiber foods.

Abstract:Abstract:［Objective］ To evaluate and compare the immunoprotection between a meq-deleted Marek s disease virus (MDV) and CVI988/Rispens against MDV very virulent strain GX0101．［Methods］ In total 120 one-day-old SPF chickens were divided into 4 groups (30 each) and kept in 5 isolators with positive pressure-filtered air．At 1 day of age，2000 PFU of SC9-1 was inoculated subcutaneously into each bird in group 1;2000 PFU of commercial vaccine CVI988/Rispens was inoculated subcutaneously into each bird in group 2．No viral challenge was made in group 3 and 4 as controls．Five days later chickens in group 1，2，3 were challenged intra-abdominally with 2000 PFU of very virulent MDV strain GX0101．During 90 days after challenge，all dead birds were recorded and checked for necropsy．The tumorsuspected tissues were examined by histopathological biopsy．The antibody titers induced by AIV and NDV vaccination and propagation dynamics of MDV GX0101 were detected．At the same time，parallel tests were performed on Hy-Line Brown chickens containing MDV maternal antibody．［Results］ SC9-1 stain provided 100% protective efficiency against very virulent GX0101 challenge in SPF and Hy-Line Brown chickens．CVI988/Rispens provided 86.7% protective efficiency against very virulent GX0101 challenge in SPF chickens and 93% in Hy-Line Brown chickens．Challenge with GX0101 caused 53.3% mortality and 16.7% of birds with gross tumors in SPF chickens while there was 36.7% mortality and 16.7% of birds with gross tumors in Hy-Line Brown chickens，and there was no tumor lesion in histopathological biopsy in control group．The results of qPCR demonstrated that the copies of GX0101 viral genomes in SC9-1 vaccinated chickens was lower than CVI988/Rispens vaccinated chickens in lymphocyte and feather follicle DNA．The results of hemagglutination inhibition test demonstrated that antibody titers of AIV and NDV was higher in SC9-1 vaccinated chickens than that in CVI988/Rispens vaccinated chickens．［Conclusion］SC9-1 stains immunoprotection against MDV is more effective than CVI988/Rispens strain s both in SPF chickens and commercial Hy-Line Brown chickens containing maternal antibody．

Abstract:Abstract:［Objective］ To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B.anthracis．［Methods］We downloaded the whole genome sequence of 6 B．anthracis strains and extracted the CRISPR sites．We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B．anthracis strains by PCR and sequenced these fragments．In order to reveal the polymorphism of CRISPR in B．anthracis，wealigned all the extracted sequences and sequenced results by local blasting．At the same time，we also analyzed the CRISPR sites in B．cereus and B．thuringiensis．［Results］We did not find any polymorphism of CRISPR in B．anthracis.［Conclusion］The molecular typing approach based on CRISPR polymorphism is not suitable for B．anthracis，but it is possible for us to distinguish B．anthracis from B．cereus and B．thuringiensis.

Abstract:Abstract:［Objective］Using molecular ecology methods，we screened non-hydrocarbon carbon sources suitable for growth of syntrophic hydrocarbon-degrading Syntrophus sp.［Methods］ The acclimated methanogenic hexadecane-degrading consortium M82 was subcultured with dodecanedioic acid，tetradecanedioic acid，hexadecanoic acid，propionate and lactate． PCR-DGGE and qPCＲ were used to analyze the abundance and quantity of syntrophaceae using different carbon sources．The T-RFLP was applied to analyze archaeal community．［Results］ The consortium M82 could grow and produce methane using a variety of fatty acids that also resulted in the change in bacterial microbial community structure．Syntrophaceae bacterial stripe was obviously detected in the culture added additional dodecanedioic acid and tetradecanedioic acid．Furthermore，the results show that the logarithmic abundance of Syntrophaceae was 7.4 and 7.6 in per milliliter culture in the two enrichment cultures respectively，which were 2－3 units higher than these in other cultures．The archaeal community structure was mainly composed of acetoclastic methanogens Methanosaeta and hydrogenotrophic methanogens Methanoculleus in all culture．［Conclusion］ Syntrophus sp．can use non-hydrocarbon carbon source ( dodecanedioic acid and tetradecanedioic acid) as substrate to grow，which provides valuable information to isolate syntrophic hydrocarbon bacteria，and reveal the molecular mechanism of syntrophic hydrocarbon degradation．

Abstract:Abstract:［Objective］Ulcer disease is one of the most serious diseases and a common problem in various stages marine culture including Epinephelus coioides culture of southern China．The isolation and identification of pathogenic bacteria from E.coioides will be useful for monitoring of drug resistance and controlling the outbreak and spread of ulcer disease in E.coioides．The purpose of this study was to characterize the pathogen of E.coioides．［Methods］ The pathogenic bacteria separated from the liver and kidney of diseased fish were identified through pure culture，artificial infection，automatic tests in bacteriology automatic identification，drug sensitive tests，morphometry，and physiological and biochemical determination.［Results］ The strains were characterized and identified as Vibrio alginolyticus． Two strain were selected for virulence tests and all the moribund/dead fish exhibited ulcer disease as that observed in natural outbreak．Drug sensitive tests show that V．alginolyticus was highly resistant to 3 agents including penicillin，whereas sensitive to 5 agents including chloromycetin．Histopathological changes were mainly shown as cell degeneration and necrosis of gill，liver and kidney，and alterative inflammation as a result of inflammatory cell infiltration in the diseased tissue．［Conclusion］ The biochemical，physiological tests confirm that V．alginolyticus is the pathogen causing E．coioides vibriosis．The multi-drug resistance among V．alginolyticus suggests strengthened monitoring of outbreaks of V．alginolyticus caused disease in E．coioides culture.