Abstract:Abstract:Noroviruses are regarded as the leading cause of epidemic gastroenteritis all over the world，with a wealth of genetic diversity．And GⅡ.4 is regarded as the predominant strain，which has constantly infected humans attributed to a fast rate of evolution in the past two ecades． With the discovery of virus binding-receptor and understanding of immune specificity，the research of evolution mechanism of the virus has been continuously developed recently． It has been hypothesized that the receptor switching and antigenic drift maintains GII． 4 persistence in human populations．Also the RNA viruses，high mutation rate and the limited space of genomic variation have also affected the direction of norovirus evolution.

Abstract:Abstract:［Objective］This study aimed to investigate the genetic diversity of 174 isolates of symbiotic bacteria associated with Acacia melanoxylon obtained from 15 sampling sites in Guangdong，Fujian and Jiangxi provinces of China.［Methods］The 16S rDNA restriction fragment length polymorphism (RFLP) and phylogenetic analyses of the 16S rDNA and housekeeping genes (recA，glnII and atpD).［Results］In the 16S rDNA PCR-RFLP analysis，9 rDNA types were identified among the 174 isolates; Phylogenetic analyses based on 16S rDNA and housekeeping gene sequences indicated that 34 representative isolates belonged to the genus Bradyrhizobium，Rhizobium，Mesorhizobium in Alpha-Proteobacteria，and the most closely related strains are Bradyrhizobium liaoningense，Bradyrhizobium betae，Bradyrhizobium cytisi，Rhizobium multihospitium and Mesorhizobium plurifarium.［Conclusion］All of the isolates could be identified to general，and Bradyrhizobium，Rhizobium or Mesorhizobium could be the dominant microsymbiont． The microsymbionts associated with Acacia melanoxylon showed relative genetic diversity.

Abstract:Abstract:［Objective］To establish a stable transformation system of the thermophilic fungus Thermomyces lanuginosus for its insertional mutagenesis.［Methods］ Agrobacterium tumefaciens-mediated transformation (ATMT) was applied to establish transformation system of T.lanuginosus. Southern blotting of hph gene and cloning of transforming DNA (TDNA) flanking sequences were used to determine insert number and site of T-DNA in the fungal genome，respectively．［Results］A reliable transformation method is established for T．lanuginosus．Specifically，pre-germinating spores of T.lanuginosus used at co-cultivated period was a prerequisite．T.lanuginosus germinating spores co-cultivated with Agrobacterium tumefaciens at 28℃ for 48 h achieved the highest transformation efficiency．Addition of Acetosyringone (AS) during pre-culture of A．tumefaciens and co-cultivation of T．lanuginosus germinating spores with A．tumefaciens was essentially required，and the best results were obtained with AS at the concentration of 500μM．Southern blotting analysis showed that majority of transformants (79.2%) contained a single insertion of T-DNA.Thermal asymmetric interlaced PCR (TAIL-PCR) analysis showed random insertion of T-DNA in the fungal genome．Using the transformation system，some stable phenotypic mutants of T．lanuginosus were obtained．［Conclusion］We report，for the first time，a simple and efficient method for transforming T. lanuginosus by using ATMT．This approach provides a tool for insertional mutagenesis gene tagging in this thermophilic fungus.

Abstract:Abstract:［Objective］ To investigate function of transporter genes fscTI and fscTII in the biosynthetic gene cluster of candicidin/FR-008． ［Methods］We constructed a plasmid pJTU4137 for disruption of transporter genes fscTI and fscTII by conjugation and homologous recombinant． The transporter genes were also PCR amplified and cloned into the high-copy plasmid pJTU1278 for overexpression in strain ZYJ-6 derived from Streptomyces sp. FR-008.［Results］The disruption mutant LX10 was unable to produce candicidin and its analogues． Overexpression of FscTI and FscTII in ZYJ-6 caused a 1.5-fold increase in FR-008-III production compared with the control.［Conclusion］We confirmed that fscTI and fscTII are function as ATP dependent ATP binding cassetle (ABC) transporters in the biosynthetic gene cluster of FR-008. Furthermore，a positive example was provided for improving antibiotic production in other polyene producing strains based on the results that overexpression of fscTI and fscTI increased candicidin production.

Abstract:Abstract:［Objective］Mycobacterium tuberculosis can survive inside host cells for a long time． To elucidate the acid adaptation mechanism mediated by the asparagine (Asn) metabolic pathway，the activity of asparaginase (AnsA) and growth of mycobacteria were characterized． ［Methods］The nonpathogenic M.bovis BCG was used as a model strain to characterize AnsA function. The recombinant AnsA was further expressed in E.coli，purified by Co2 + charged resin，and analyzed by enzymatic method．M．bovis BCG mutant (DansA) was constructed by homologous recombination．The growth and ammonia generation by M．bovis BCG wild type and mutant were evaluated in acidic medium．［Results］Ammonia was detected in the presence of AnsA and Asn．Deletion of ansA significantly delayed the growth of M．bovis BCG for 10 days in acidic medium． Asn was used by M．bovis BCG to produce ammonia，increasing pH．［Conclusion］The acid adaptation of M．bovis BCG was mediated by ammonia，the metabolic product of asparagine．This provides new clue to uncover the survival mechanism of M．tuberculosis inside the acidic niche environment of host macrophage．

Abstract:Abstract:［Objective］ To determine the secondary metabolites production in mycelia of Paecilomyces militaris．［Methods］Mycelia were cultured in plates with sabouraud dextrose agar yeast medium at 25℃ for 9 days． Sampling was done every day from the second to the ninth day． The secondary metabolites in the mycelia of Paecilomyces militaris were extracted with either methanol or ethyl acetate．The extracts were blended and analyzed by liquid chromatography-mass spectrometry (LC-MS)．LC-MS data were collected and analyzed by MetaboAnalyst software．［Result］ Principal component analysis indicates different secondary metabolites accumulation with incubation times． Hierarchical clustering analysis shows that the metabolic process of cationic compounds such as alkaloids，peptides and nucleosides can be divided into three stages，and that the metabolic process of anionic compounds such as organic acids and saccharides can be divided into two stages． Metabolites difference and heat map analysis show that:(1) The number of metabolites with significant increased contents was raised significantly in mycelia of Paecilomyces militaris on the second and third incubation days．The main species with increased contents were esters and their hydrolized products，destruxin B，variotin and some unidentified nitrogin contained compounds．(2) The number of metabolites with significant raised contents was decreased significantly on the fourth and fifth incubation days．The main species with increased contents were ophiocordin and destruxin A．(3) Apart from peptide antibiotics such as several beauverolides，the content increased metabolites included also several organic acids，amino acids，rhamnose，trehalose，cerebroside and riboflavine during the sixth to ninth incubation days．［Conclusion］ The secondary metabolites in mycelia of Paecilomyces militaris were related significantly to the incubation time.

Abstract:Abstract:［Objective］The aim of the present study was to investigate the soil bacterial diversity of pine forest (PF)，pine-broadleaf mixed forest (MF) and monsoon evergreen broadleaf forest (MEBF)，the typical forest types that represent early，middle and late successional stage forests in Dinghushan，respectively． The results obtained will also provide information for further examination of the relationship between the soil bacterial diversity and ecological function of the forests． ［Method］ Three total DNA samples were extracted directly from soil samples collected from PF，MF and MEBF，and then the 16S rDNA sequences were PCR amplified and the libraries were constructed，respectively． For each of the three libraries constructed，150 positive clones were picked randomly and the inserted 16S rDNA were sequenced．The soil bacterial diversity of the forests was analyzed by Mothur based on the sequences obtained． ［Result］A total of 122，118 and 120 valid 16S rDNA sequences were obtained from PF，MF and MEBF，which represented 70，64 and 72 operational taxonomic units (OTUs，definition at a level of 97% similarity)，respectively． Bacteria belonging to 8 phyla were identified． Among them，Acidobacteria accounted for 53. 3% ，67. 8% and 60% ，while Proteobacteria took up 29. 5% ，20. 3% and 32. 5% in PF，MF and MEBF，respectively． The other bacterial phyla identified each accounted for less than 10% ． The bacterial community structure differed significantly at species level among three soil samples (P＜0.05) with the percentages of the shared OTUs between any two soil samples lower than 25% ． MEBF had the highest Chao index (414. 2)，Shannon index (3. 90) and the lowest Simpson dominance index (0.0249)．［Conclusion］The soil bacterial community structure differed significantly at species level among PF，MF and MEBF in Dinghushan，while they have a similar structure at phyla or class levels with Acidobacteria predominated followed by Proteobacteria．

Abstract:Abstract:［Objective］To elucidate the arsenic metabolic pathway of purple nonsulfur bacteria (PNSB).［Methods］We investigated the distribution within their genomes，organization，composition，arrangement，core genes and coding proteins of arsenic gene clusters found in complete genome from 17 strains of PNSB by comparing the genomes analysis，and studied the arsenic metabolism in 3 members of PNSB under anaerobic conditions by UV-Vis and HPLC-ICP-MS．［Results］ Arsenate reduction and arsenite methylation pathways mediated by ars operon are the dominating arsenic metabolic processes． The arsenic gene clusters differ vastly in composition and arrangement．Some members of PNSB evolved two independently families of arsenate reduction genes (arsC). The cells of Rhodopseudomonas palustris CQV97，Rhodobacter azotoformans 134K20 and Rhodobacter capsulatus XJ-1 could reduce As (Ⅴ) to As (Ⅲ)，whereas As (Ⅲ) could not be transformed back to As (Ⅴ). Higher concentration phosphate competitively inhibited arsenate toxicity to cells.［Conclusion］Our investigations shed light on the evolution and functional implications in arsenic gene clusters of PNSB，and support the notion that arsenate reduction and arsenite methylation appears to be the dominant process in PNSB.

Abstract:Abstract:［Objective］To investigate specific immune responses elicited by a recombinant Listeria monocytogenes strain LM4△hly:: E7 and assess protective effect in C57BL/6 mice.［Methods］ C57BL/6 mice were intraperitoneally immunized with LM4△hly::E7 at 1-week intervals．After the second immunization，cellular immunity elicited by this recombinant L．monocytogenes strain was analyzed via an ELISPOT assay，Cytotoxic T lymphocytes (CTL) measurement assay and analysis of effector T cells proportion in the splenocytes．Also，the serum antibodies against HPV16 E7 protein were determined in an ELISA assay．Finally，protective effect was assessed against the challenge with TC-1 tumor cells.［Results］The immune responses elicited by LM4△hly::E7 were biased towards Th1 type in the ELISPOT assay．Also，LM4△hly::E7 was able to induce E7-specific CTL activity，with average specific lysis of 72%，which was highly significant difference compared with the controls (P＜0.01)．Moreover，the proportion of effector T cells in the spleens were increased in mice immunized with recombinant L．monocytogenes strain (P＜0.05). The titer of E7-specific antibodies in mice immunized with LM4△hly::E7 was 1:400 in the ELISA assay． Furthermore，immunization with LM △hly::E7 protected all mice against the lethal tumor cell challenge．［Conclusion］ The data suggest that attenuated Listeria monocytogenes delivering HPV16 E7 antigen could induce both E7-specific cell mediated immunity and humoral immunity，and had a protective effect against challenge with TC-1 tumor cells.

Abstract:Abstract:［Objective］ The signaling lymphocyte activation molecule (SLAM，also known as CD150)，is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM(rSLAM) to efficiently isolate CDV from pathological samples.［Methods］ A eukaryotic expression plasmid，pIRES2-EGFPrSLAMhis，containing rSLAM gene fused with six histidine-coding sequence，EGFP gene，and neomycin resistance gene was constructed. After transfection with the plasmid，a stable cell line，Vero-rSLAM，was screened from Vero cells with the identification of EGFP reporter and G418 resistance． Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation． Foxes and raccoon dogs were inoculated subcutaneously LN (10) f1 strain with 4×102. 39 TCID50 dose to evaluate pathogenicity of CDV isolations.［Results］The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay．Three CDV strains were isolated successfully in Vero-rSLAM cells 36－48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper． By contrast，no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation．Infected foxes and raccoon dogs with LN(10) f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge.［Conclusion］Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

Abstract:Abstract:［Objective］To clone and express fan operon gene clusters of K99 fimbriae in enterotoxigenic Escherichia coli (ETEC) in vitro，and study the activity of the recombinant E.coli expressing K99 fimbriae.［Methods］K99 fimbriae gene clusters were amplified by long-PCR method，using the genomic DNA of K99-fimbriae E.coli C8307 as the DNA template. The 5.7Kb PCR products were inserted into expressing vector pBR322 with restriction endonuclease，then positive clones were screened． The positive recombinant plasmid was transformed into non-fimbriae E.coli SE5000 strains，and pBR322 plasmid was also transformed into SE5000 for negative control strain.［Results］The recombination E.coli expressing K99 fimbriae was tested with agglutination assay，using monoclonal antibody serum and brush border vesicles from the piglet small intestinal epithelia cells. The expressed fimbriae on the surface of the recombinant E.coli SE5000 were observed by transmissible electromicroscope．Heat extraction method was employed to isolate and purify K99 fimbriae，which was exerted SDS-PAGE，and 18.5 kDa protein band was detected. The mouse sera produced from recombinant fimbriae was used to test K99-fimbriae strains C83907，C83914，C83260 with positive agglutination results， while negative results were found with E． coli contain other kinds of fimbriae． The assays of SDS-PAGE，Western blot，agglutination assay were used to evaluate antigenicity and biologic activity between C83907 and recombinant strain．Adhesion test with HeLa cell line demonstrated the recombinant strain and wild type have the similar adherence ability，and this adhesion can be inhibited with mouse serum containing polyclonal antibody against recombinant K99 fimbriae.［Conclusion］This study has laid a good foundation for further study on bioactivity of K99.

Abstract:Abstract:［Objective］We studied nonribosomal peptides synthetases (NRPSs) gene clusters and the core module of NRPSs in Pseudoalteromonas sp． NJ631 using genome mining approach.［Methods］The genome of Pseudoalteromonas sp. NJ631 was constructed by the next genome sequencing (NGS) technology. We adopted an online available software called NRPSPKS knowledgebase to identify potential NRPSs gene clusters within genes involved in the biosynthesis of secondary metabolite of Pseudoalteromonas sp. NJ631. The genes encoding adenylation(A) domains，the core module of NRPSs，were collected and analyzed using genome mining method.［Results］We identified three typical NRPS gene clusters comprising three ORFs which encode six continuous modular NRPSs. The result of genome mining indicates that genome of Pseudoalteromonas sp. NJ631 contains 38 A domain genes which show 60% similarity below to their closest relatives. The substrate of these A domains was predicted to specifically bind 18 types of amino acids using the specificity-conferring selection rule.［Conclusion］This is the first reported on the systematic screening and analysis of NRPSs gene clusters and A domains in genus Pseudoalteromonas，suggesting that the genus Pseudoalteromonas possesses a vast array of secondary metabolite biosynthesis genes that were previously found mostly in actinomycetes and fungi. The information on secondary metabolite genes from Pseudoalteromonas sp. NJ631 will facilitate us to isolate novel nonribosomal peptides.