Abstract:Abstract:Autophagy in intracellular bacterial infection is important． Host cells can use autophagy to eliminate invading pathogens． Meanwhile，autophagy can be exploited，modified or interfered by some pathogens to benefit their survival or proliferation． We reviewed the effect of autophagy on intracellular bacterial infection and discussed our study on the relationship between autophagy and Salmonella infection，which can help provide the theoretical basis for controlling pathogenic infection through regulating and exploiting autophagic pathway．

Abstract:Abstract:［Objective］To analyze the sequences and secondary structure of group-I introns in 18S rDNA of Cenococcum geophilum，and to study the factors effecting Cg genetic diversity． ［Method］The 3' end region of 18S rDNA were amplified from twenty-three Cg isolates，and the PCR fragments from thirteen isolates were sequenced． The Neighbor-Joining phylogenetic analyses were performed using MAGE version 4. 0，and the secondary structures of the Cg introns were predicted using Mfold． ［Result］The results showed that fourteen out of nineteen Cg isolates originated in China had group-I introns in 18S rDNA． Their sequences，ranging in size from 488 to 590 nucleotides，were from 92. 3% to 100% similar to each other from our data and GenBank data． The 5' end sequences of the introns were conserved，while the 3' end sequences were variable． The analysis of secondary structure indicated all the introns from forty-five Cg isolates contained ten pairing regions (P1－P10)． The P5 region was composed of P5，P5a，P5b，P5c and P5d，and there were two extra pairing stems (P9. 1、P9. 2) besides the 3' end of P9． ［Conclusion］It was suggested that Cg had rich genetic diversity，and we did not found the relationship between Cg genetic variation and its geographical and host origination．

Abstract:Abstract:Cellulosilyticum ruminicola H1 is an anaerobic cellulolytic bacterium isolated from the rumen content of yak．Previously，we found that strain H1 lost growth on filter paper cellulose after several subcultures．The growth on cellulose cannot be restored until a few transfers in the cellobiose-containing medium． This is contrary to the knowledge that microbial cellulases synthesis is induced by the substrate while repressed by the metabolites，e.g.cellobiose，but provides a hint of cell-density mediated cellulase synthesis．［Objective］ The study aimed to test if cell density regulation mechanism involved in the cellulase synthesis by Cellulosilyticum ruminicola H1．［Methods］By using enzyme assays and real-time PCR quantification，we investigated cellulase activities and the gene transcript levels in the high- and lowcellmass cultures of strain H1． We also determined the elevation of endoglucanase and cellobiohydrolase activities and the gene expression in the low-cellmass culture upon addition of the high-cellmass spent culture．［Results］ Both endoglucanase and cellobiohydrolase of strain H1 were detected 3 to 10 times higher in the high cellmass culture than in the lower cellmass culture either in enzymatic activity or the gene transcript abundance，thus confirming the cell densitymediated cellulase synthesis．［Conclusion］This study demonstrates that cell density regulation is involved in cellulases synthesis by Cellulosilyticum ruminicola H1．

Abstract:Abstract:［Objective］We studied the influence of spoIIID gene deletion on the activity of cry1Ac，cry3A，cry4A and cry8E gene promoters in Bacillus thuringiensis and compared the activity among these promoters in spoIIID mutant (HD-!SpoIIID)． ［Methods］We constructed 4 promoter fusions with lacZ gene and transformed them into wild-type strain HD-73 and HD-!SpoIIID to analyze their transcriptional activity． We constructed a spoIIID gene mutant (HD － -!SpoIIID)with deletion of the cry1Ac-harboring native plasmid based on HD-!SpoIIID strain． We constructed four promoter fusions with cry1Ac gene and transformed them into HD-ΔSpoIIID and HD － -ΔSpoIIID to perform Cry protein quantization and bioassay． ［Results］By Beta-galactosidase assay we found that the activities of the four promoters were，in decreasing order，Pcry8E ＞ Pcry1A ＞ Pcry4A ＞ Pcry3A in both HD-73 and HD-ΔSpoIIID strains． The deletion of spoIIID had no effect on transcriptional activity of Pcry1Ac and Pcry8E． The transcriptional activity of Pcry3A in HD-ΔSpoIIID was slightly higher than that in HD-73． The transcriptional activity of Pcry4A in HD-ΔSpoIIID was decreased compared to HD-73． The Cry1Ac protein production directed by Pcry1Ac was as much as Pcry8E in HD-ΔSpoIIID and higher than that by Pcry4A and Pcry3A in accordance with the bioassay result． ［Conclusion］The cry8E gene promoter is the strongest promoter among four promoters in spoIIID gene mutant at transcriptional level． The Cry1Ac protein production directed by Pcry1Ac is almost equal to that by Pcry8E．

Abstract:Abstract:［Objective］The aim of this study was to screen microorganisms that could degrade rice straw.［Methods］We used selective medium to screen strains and determined straw fracture tension strength，weight loss，lignocellulose decomposition rate and extracellular enzyme activity as re-screening methods after 10 days shake flask culture.［Results］ We isolated two antinomycetes (A3 and A6)，the highest cellulose enzyme activity of holoenzyme，β-Glucosidase，endonuclease and exonclease for A3 were 12. 84，6. 23，24. 56and 14. 00 U/mL，and for A6 12. 85，6. 53，17. 80 and 18.80U/mL．The hemicelluloses enzyme activity was 83. 05 for A3 and 52. 98 U/mL for A6. Both strains belonged to Streptomyces． With 10 days' treatment，inoculated straws showed a decrease of straw fracture tension strength by 62.67% (A3) and 66. 67% (A6)，while weight loss of straw was 31. 50% (A3) and 35. 83% (A6)． A3's decomposition rate of cellulose，hemicellulose and lignin was 38. 73%，33. 16% and 20. 68%，and 47. 69%，28. 64% and 22. 59% for A6．［Conclusion］Antinomycetes A3 and A6 could degrad cellulose，hemicellulose and lignin.

Abstract:Abstract:［Objective］To increase the leavening ability in the lean dough，the maltose utilization ability of baker's yeast was enhanced． ［Methods］A 1. 7kb PGK1 promoter and terminator were ligated and inserted into vector Yep-C to give the expression plasmid named Yep-CP． Then a 1.7kb DNA fragment containing the open reading frame and terminator of mal62 gene was amplified from Saccharomyces cerevisiae BY-14 by PCR，and inserted into Yep-CP to generate recombinant plasmid Yep-CPM. To express mal62 gene properly in S. cerevisiae，the recombinant expression plasmids Yep-CPM with copper resistance gene as the selection marker for yeast transformation were introduced into S．cerevisiae BY-14． The resulting yeast transformant BYCPM was screened on YEPD with 4 mmol/L CuSO4 and identified by colony PCR．Target protein was detected by qRT-PCR，and the enzyme activities and the leavening ability of the recombinant strain BYCPM were determined to confirm whether functional expression was achieved． ［Results］The maximum maltase activity of recombinant strain BYCPM was 15%－52% higher than that of control strain BY-C.Leavening ability and specific leavening ability of recombinant strain BYCPM were 40% and 5.6% higher than that of the control strain BY-C，respectively． ［Conclusion］The mal62-overexpression was an effective way of increasing the maltase activity and the ability of glucose depression in the baker's yeast.Moreover，recombinant strain BYCPM could further enhance the leavening ability and produce more gas while consume lesser carbon source than the control strain.

Abstract:Abstract:［Objective］To search for structurally novel and biologically active compounds from the secondary metabolites of halotolerant fungi from the Yellow River Delta area．［Methods］We screened halotolerant fungi with rich chemical diversity and antitumor or antimicrobial activity by means of integrated chemical and biological method． We cultured halotolerant fungi under different conditions at first． Then we investigated the chemical diversity and the bioactivity of the EtOAc extracts of the fermentation broth by HPLC and TLC，and cytotoxic assay or antimicrobial assay． We selected Penicillium chrysogenum HK14-01 to further study for the large yield，producing alkaloids and cytotoxicity on P388 cells in YMDP culture medium containing 10% NaCl． We fermented P． chrysogenum HK14-01 on a large scale; we isolated and purified the compounds by column chromatography over silica gel，Sephadex LH-20，and semipreparative HPLC; and we identified the structures by spectroscopic analysis，X-ray diffraction (Mo-Kα)，CD spectra and the time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculation． ［Results］We isolated and identified a halotolerant fungal strain，P． chrysogenum HK14-01，from the sediments collected in the Yellow River Delta area． From the fermentation broth of P. chrysogenum HK14-01，we isolated and identified eight compounds，i.e. (2S，3R)-oxaline (1，a major product)，(3R，4R)-3，4，8-trihydroxy-3，4-dihydronaphthalen-1(2H)-one (2)，(Z)-N-(4-hydroxy styryl) formamide (3)，(E)-N-(4- hydroxystyryl) formamide (4)，emodin (5)，4-(2- hydroxyethyl) benzene-1，2-diol (6)，methyl 2-( 4- hydroxyphenyl ) acetate (7)，and 2-(4-hydroxyphenyl ) acetonitrile (8).［Conclusion］ Bioactive compounds can be obtained from the secondary metabolites of halotolerant microorganisms from the Yellow River Delta area．

Abstract:Abstract:［Objective］To isolate and identify the cellulose-producing bacterium from fresh feces of healthy giant pandas，and characterize its cellulase production． ［Methods］A strain with high activity of cellulase was isolated and purified by carboxymethyl cellulose-Na (CMC-Na) medium． The morphological，physiological and biochemical characteristics and 16S rDNA gene were analyzed to identify the taxonomic position of the strain． Meanwhile，its cellulase producing conditions and degradation of several cellulose substrates were studied． ［Results］A cellulose-producing strain P2 was obtained． Strain P2 is a gram-positive and aerobic bacterium． Its growth temperature ranges from 20 to 50℃ (optimum at 37℃) and pH 6.0 to 9.0 (optimum at 7.0)，and NaCl concentration at 0%－15% (optimum at 2.0%)． It took 24 h to reach the peak of cellulase production． Phylogenetic analysis based on 16S rDNA sequence showed that strain P2 is most closely related to the Bacillus amyloliquefaciens NBRC15535 with the similarity of 99. 66%． Strain P2 posses different abilities to degrade four different cellulose substrates including filter paper，absorbent cotton，straw，and bamboo fiber． During the degradation，it shows different enzymatic activity curves with endoglucanases，exoglucanases，β-glucosidases and cellulase． ［Conclusion］An aerobic cellulolytic bacterium was isolated from feces of giant pandas for the first time，and was identified as Bacillus amyloliquefaciens． Due to its ability to degrade the cellulose materials with different structures，strain P2 could be used in the further study on the digestion mechanism of bamboo of giant panda．

Abstract:Abstract:［Objective］To identify the interaction between influenza A virus PB1-F2 and human modulator of apoptosis 1 (MOAP-1)．［Methods］The recombinant plasmid pACT2-MOAP-1 was constructed and co-transformed into yeast AHl09 with pGBKT7-PB1-F2． The growth of the co-transformants on quadruple dropout medium and β-galactosidase activity of the reporter gene were tested． We further confirmed the interaction of cellular protein MOAP-1 and PB1-F2 by glutathione S-transferase(GST)pull-down and co-immunoprecipitation (Co-IP) assays． In addition，we investigated the effect of PB1-F2 on MOAP-1 protein level by Western blot．［Results］The results of yeast two-hybrid assay showed that MOAP-1 specifically interacted with PB1-F2 in yeast cells． Furthermore，the binding of MOAP-1 with PB1-F2 was demonstrated by glutathione S-transferase pull-down and Co-IP assays． PB1-F2 could upregulate exogenous MOAP-1 protein level．［Conclusion］These results suggested that influenza virus PB1-F2 interacted with MOAP-1 and it might be involved in the regulation of cell growth and apoptosis via association with MOAP-1．

Abstract:Abstract:［Objective］Aflatoxins are toxic and carcinogenic fungal metabolites． The aim of this research was to isolate aflatoxin B1(AFB1)-degrading bacteria． ［Methods］Samples from various sources were screened by using coumarin as the sole carbon source． The strains that could grow in the medium with coumarin carbon source were detected for their degradation of AFB1 ability by addition of AFB1(2.5μg/mL) to the cultured broth． Among the positive strains，F4 strain with the highest activities was identified according to its morphological，physiological and biochemical properties together with phylogenetic analysis of its 16S rRNA sequence． The effects of degradation conditions such as bacterial cell concentration，pH，temperature，metal ions on the degrading AFB1 were investigated． ［Results］Ten isolates showed good AFB1 reduction activity and they could grow well on coumarin carbon source． Strain F4，obtained from Budorcas taxicolor feces could reduce AFB1 by 90. 03% after incubation in the liquid medium at 37℃ for 72 h． F4 was preliminary identified to be Pseudomonas stutzeri with morphology，physiological and biochemical properties and 16S rRNA gene sequence． The active degrading component existed in the cell of F4，and the degrading activity was interrelated with cell concentration，temperature，pH and metal ions． ［Conclusion］ An AFB1-degrading strain F4 was isolated from animal feces and identified as Pseudomonas stutzeri． The activive component of AFB1 degradation was mainly in the cell of F4.

Abstract:Abstract:［Objective］To investigate the antiviral effect of the flavonoid compound flavopiridol on influenza A virus and explore its antiviral mechanism． ［Methods］The A549 or Madin-Darby canine kidney (MDCK) cells were infected with influenza A virus A/WSN/33 and treated with flavopiridol． The viral proteins were determined by immunolotting and immunofluorescence． The virus titer was measured by plaque assay． To verify whether the activity of host RNA polymerase Ⅱ was affected by flavopiridol，the phosphorylation status of RNA polymerase Ⅱ CTD domain was analyzed by immunoblotting with phosphor-specific antibody． The amount of viral mRNA，vRNA and cRNA was measured by reverse transcription and PCR． ［Results］The amount of viral proteins was significantly decreased and the titer of virus was greatly reduced in cells treated with flavopiridol． Further analysis showed that the phosphorylation of Ser-2 in the heptad repeat of the CTD domain in RNA polymeraseⅡ was decreased in falvopiridol treated cell． This result indicated that the transcription elongation activity of RNA pol II was impaired upon treatment with flavopiridol． Then we found that the amount of viral vRNA was significantly decreased in flavopiridol treated cells while only moderate decrease of mRNA was observed and almost no reduction of cRNA was detected． ［Conclusion］Flavopiridol can greatly suppress the replication of influenza virus． We propose that the inhibition of the transcription elongation activity of host RNA polymeraseⅡ would cause the decrease of viral mRNA transcription．

Abstract:Abstract:［Objective］The vast majority of microbial resources has not been exploited and utilized because more than 99% of microorganisms in soil cannot be cultured by conventional means． The quantity and quality of environmental DNA (eDNA) are the most important issues in metagenomic study． Here we set up an optimized method for high quality soil microbial DNA isolation，which can be applied for microbial diversity study and metagenomic library construction containing large eDNA inserts． ［Methods］After comprehensive comparison of strengths and weaknesses of the commonly used methods for microbial DNA isolation，we proposed a new method by optimizing several key procedures of isolation，including combination of sodium dodecyl sulfate ( SDS)-hexadecyltrimethylammonium bromide (CTAB) and lysozyme to lysis cells， usage of chloroform in stead of phenol to remove protein contamination， filtration with the polyvinylpolipyrrolidone (PVPP) column to purify DNA． Using three different soil samples，we compared the optimized method with three reported methods in the literature by analyzing soil eDNA yield，rate of purity，size and ability of PCR amplification． ［Results］The quality of soil eDNA isolated by the optimized method was significantly improved． We obtained 95 μg DNA per gram of soil． OD A260 /A280 and A260 /A230 ratios of DNA were much closer to the ideal level． More desired PCR product was amplified and maximum size of eDNA reached 100 kb． ［Conclusion］High quality and quantity of soil microbial DNA can be isolated by the optimized method we established，which provides a powerful tool for metagenomic research to exploit uncultured microbial resources in soil.

Abstract:Abstract:［Objective］We developed a homologous recombination system to make marker-free mutants in Mtb in an easy screening way． ［Methods］The plasmid pSL002 harboring recE and recT-like protein gp60 and gp61 was transformed into wt Mtb in order to increase the recombing efficiency． The two homologous arms of target gene were amplified and cloned into pSL001． The purified homologous arms-loxP-gfp-hyg-loxP fragments were further transformed into Mtb (with pSL002 inside) competent cells to obtain the double cross over (DCO) mutants． The plasmid pSL003 was transformed into DCO competent cells to excise the two loxP sites flanked region． The plasmid pSL002 and pSL003 were removed via the counter selection marker sacB． ［Results］An efficient homologous recombination system was developed． Three different marker free mutants: Rv1364c phosphatase domain，PstP extracellular domain and PstP transmembrane domain were created by the system developed in this study． The efficiency to obtain DCO was varying from 25% to 62. 5%，while the efficiency from DCO to KO was close to 100%． The gfp reporter was used to screen SCO，DCO and KO． ［Conclusion］The homologous system shortens the complicated screening process to 3 month in Mtb． This is the fastest allelic exchange system reported so far，providing novel deletion strategy to the repertoire of mycobacterial genetic tools for constructing unmarked mutations．

Abstract:Abstract:［Objective］To identify a thermophilic bacterium from horse manure to degrade cellulose efficiently，and to enrich microbial resources producing cellulolytic ethanol by co-culturing with thermophilic ethanol producing bacterium．［Methods］We used Hungate anaerobic technique to isolate a strain named as HCp from horse manure mixed culture; its phylogeny was identified through 16S rDNA sequencing． Enzymatic assays were determined using DNS method． ［Results］ The isolated HCp cells were straight with rods size of(0.35－0.50)μm×(2.42－6.40)μm，in the form of single or paring． This strain belongs to a strictly anaerobic Gram-negative bacterium，it is able to form spores，shows motile ability and resistance to neomycin． The strain could degrade filter paper cellulose，cellulose powder，microcrystalline cellulose，cotton wool，rice straw and gelatin，and it was also able to utilize abundant saccharides as substrates such as cellobiose，glucose，xylose，xylan，raffinose，maltose，sorbose，fructose and galactose． The growth pH ranges from 6. 5 to 8. 5，temperature from 35 to 70℃ and concentration of NaCl on cellulose from 0% to 1. 0%，while the optima of pH6. 85，60℃ and 0. 2% NaCl． Under the optimal growth conditions，the filter paper cellulose degradation rate was up to 90. 40% after 10 days． The optimum temperatures for FPA，CMCase，β-glucosidase and xylanase were 70℃，70℃，70℃，and 60℃ respectively． CMCase activity was found with high thermal stability． The phylogenetic analysis based on partial 16S rDNA revealed that HCp was close to Acetivibrio cellulolyticus and A． cellulosolvens with 97. 5% sequence similarities．［Conclusion］Strain HCp is thermophilic，efficiently cellulolytic anaerobe． It is able to utilize vast substrates and produce highly thermostable enzymes． It is a potential bacterium that can be used for cellulolytic ethanol production．

Abstract:Abstract:［Objective］The LuxS /AI-2 quorum sensing (QS) system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2)． In this study，the method of biosynthesis of AI-2 was established using recombinant LuxS and Pfs of avian pathogenic Escherichia coli (APEC)，which will be benefit for future study of the role of AI-2 in APEC． ［Methods］ We investigated AI-2 production in APEC by vibrio harveyi BB170 (BB170)．Furthermore，APEC LuxS and Pfs were expressed，purified and used to investigate the production of AI-2 in vitro． LuxS and Pfs were incubated with S-ribosylhomocysteine (SAH)，the reaction was detected for the production of luminescence of BB170． ［Results］APEC can produce AI-2 by BB170 bioassay． Purified LuxS and Pfs enzymes incubated with SAH and produced 300μmol/L AI-2 in the reaction products． ［Conclusion］The results demonstrated that recombinant Pfs and LuxS synthesize AI-2 in vitro from SAH． These findings will be of benefit to future studies of the role of AI-2 in APEC．