Abstract:Abstract:The transfer of resistance gene is one of the most important causes of bacterial resistance． Recent studies reveal that stresses induce the transfer of antibiotic resistance gene through multiple mechanisms．DNA damage stresses trigger bacterial SOS response and induce the transfer of resistance gene mediated by conjugative DNA． Antibiotic stresses induce natural bacterial competence for transformation in some bacteria which lack the SOS system． In addition，our latest studies show that the general stress response regulator RpoS regulates a novel type of resistance gene transfer which is mediated by double-stranded plasmid DNA and occurs exclusively on the solid surface． In this review，we summarized recent advances in SOS dependent and independent stress-induced DNA transfer which is mediated by conjugation and transformation respectively，and the transfer of double-stranded plasmid DNA on the solid surface which is regulated by RpoS．We propose that future work should address how stresses activate the key regulators and how these regulators control the expression of gene transfer related genes．Answers to the above questions would pave the way for searching for candidate targets for controlling bacterial resistance resulted from the transfer of antibiotic genes．

Abstract:Abstract:Pullulanase and isoamylase belong to the GH13 family (glycoside hydrolase family 13) with similar sequence，catalytic mechanism and three-dimensional fold ((β/α) 8-barrel structure)．Starch debranching enzymes can hydrolyze the α-1，6-glucosidic bonds at the branch sites of starch，and improve raw material utilization and production efficiency in the starch industry．In this review，the substrate specificity，protein structure，advances and new trends in the study of microbial GH13 starch debranching enzyme were systematically introduced．In addition，some opinions on the research status and prospect for starch debranching enzyme were discussed．

Abstract:Abstract:［Objective］We isolated myxobacteria and explored their diversity from the rhizosphere soils of some medicinal plants．［Methods］ We used the helper bacteria baiting technique to isolate myxobacteria from the rhizosphere soils collected in South China Botanical Garden and Nanling National Forest Park． The myxobacteria were identified by morphological characteristics and 16S rDNA gene sequences analysis． ［Results］A total of 50 strains were isolated from 22 soil samples，which were identified into 7 genera，Myxococcus (18)，Corallococcus (11)，Cystobacter (7)，Archangium (8)，Stigmatella (1)，Chondromyces (4) and Pyxidicoccus (1)．The dominant genera were Myxococcus and Corallococcus． ［Conclusion］Environmental factors were associated with the diversity of myxobactria．Myxobacteria better adapt in high organic matter content and neutral pH environments．The strains of Myxococcus and Corallococcus had a good adaptability for different pH．Meanwhile，the dependence of the strains of Myxococcus and Cystobacter on organic carbon content was not too obvious，and they can also be found in the poor soils． Our findings provided an important scientific base for the development and utilization of myxobacteria resources．

Abstract:Abstract:［Objective］ Using the 16S rRNA，dnaA，murC and pyrG gene sequences，we identified the phylogenetic relationship among closely related Leuconostoc citreum species.［Methods］Seven Leu． citreum strains originally isolated from sourdough were characterized by PCR methods to amplify the dnaA，murC and pyrG gene sequences，which were determined to assess the suitability as phylogenetic markers．Then，we estimated the genetic distance and constructed the phylogenetic trees including 16S rRNA and above mentioned three housekeeping genes combining with published corresponding sequences.［Results］By comparing the phylogenetic trees，the topology of three housekeeping genes trees were consistent with that of 16S rRNA gene． The homology of closely related Leu． citreum species among dnaA，murC，pyrG and 16S rRNA gene sequences were different，ranged from75.5% to 97.2%，50.2% to 99.7%，65.0% to 99.8% and 98.5% 100%，respectively．［Conclusion］ The phylogenetic relationship of three housekeeping genes sequences were highly consistent with the results of 16S rRNA gene sequence，while the genetic distance of these housekeeping genes were extremely high than 16S rRNA gene． Consequently，the dnaA，murC and pyrG gene are suitable for classification and identification closely related Leu．citreum species.

Abstract:Abstract:［Objective］To elucidate the pathogenesis role of the vir region of APEC O2 strain E058.［Methods］The gene aerobactin/sitABC operon knockout mutants E058Δvir of APEC E058 strain was generated using Red recombination system． A series of pathogenicity tests including chick embryo inoculation，the competition experiment and the colonization and persistence in vivo were used to evaluate the pathogenicity of APEC E058Δvir and the wild-type strain E058.［Results］E058Δvir was similar to its parental strain E058 in the growth curves，invasion assays of HD-11 cell and in vitro competition assay． In the colonization and persistence test，the recovery colonies of E058Δvir were significantly decreased in all of the organs tested (P＜0.001).［Conclusions］These results indicate that the virulence factors encoded by aerobactin/sit operon genes were important for the pathogenesis of APEC E058.

Abstract:Abstract:［Objective］To study the transcriptional regulation mechanism of dps by OxyR in Yersinia pestis.［Methods］Total RNAs were extracted from the wide-type (WT) strain and the oxyR null mutant (ΔoxyR).Primer extension assay was used to detect the promoter activity (the amount of primer extension product) of dps in WT and that in ΔoxyR. Then，quantitative RT-PCR was done to calculate the transcriptional variation of dps between the WT and ΔoxyR．The entire promoter-proximal region of the dps gene was amplified by PCR from Y．pestis strain 201．Over-expressed His-OxyR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham)．Then，the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-OxyR to dps promoter region in vitro． Finally，Escherichia coli OxyR consensus was deployed to predict the OxyR binding site to dps promoter region.［Results］The primer extension assay detected only one transcriptional start site located at 40bp uptream of dps，whose transcript was under positive regulation by OxyR． The bioinformatics analysis indicated that His-OxyR bound to a single region from 111bp to 78bp upstream of dps.［Conclusion］The transcription of dps was directly activated by OxyR in Yersinia pestis.

Abstract:Abstract:［Objective］ A new naringinase-producing strain，JMUdb058 was identified and characterized. ［Methods］The strain was identified by morphological observation and 28S rDNA homogeneous analysis． Naringinase was identified by monitoring the hydrolysis of naringin to prunin and naringenin using a reversed phase High Performance Liquid Chromatography (HPLC)．The regulation of naringinase expression was studied by measuring naringinase activity of 11 different carbon sources and 7 nitrogen sources in shaking cultivation． The naringinase-producing capacity was investigated in both solid-state fermentation and submerged fermentation．［Results］The macro-morphology and micro-morphology of JMUdb058 corresponded to the characteristics of Aspergillus section Nigri Gams，and the 28S rDNA sequences showed homogeneity at 100% to Aspergillus aculeatus． Crude enzymes prepared by both submerged fermentation and solid-state fermentation could hydrolyze naringin to prunin and naringenin．In addition，the enzyme could remove naringin from citrus juice effectively． Carbon resources，including hesperidin，naringin，rutin and rhamnose，and organic nitrogen resources，i.e.，tryptone，soybean meal，yeast extract and corn syrup were shown to express the naringinase．The strain had an outstanding ability to yield naringinase in the solid-state fermentation，which showed an α-L-rhamnosidase activity of 5903 U/gds by HPLC，and the naringinase of 1939U/gds by HPLC and 72232 U/gds by Davis method.［Conclusion］It is the first time to report a stain of Aspergillus aculeatus can produce naringinase，carbon source containing rhamnose groups are able to induce the enzyme expression．The stain JMUdb058 is a new microorganism source for high yield of naringinase，in particularly by the solid-state fermentation.

Abstract:Abstract:［Objective］We screened and identified protease-producing bacterial strains from the Arctic，the results would help find cold-adapted protease．［Methods］In total 68 protease-producing strains were screened from the Arctic using the casein-agar plate under low temperature． All strains were classified using the 16S rRNA gene-restriction fragment length polymorphism (RFLP) and traditional phenotypic test． One strain was chosen to be the representative strain from each group respectively and identified using 16S rRNA gene sequence analysis，GenBank database Basic Local Alignment Search Tool (BLAST)，phylogenetic analysis and phenotypic features analysis．The enzymatic properties of the epresentative strains were determined．［Results］ The 68 strains belonged to 3 groups (54.41%，42.65% and 2.94%)，and strains 6，11 and 52 were the representative strains respectively．The results of the 16S rRNA gene sequence analysis showed that:Strain 11 was most closely related to Chryseobacterium scophthalmum with 98.24% sequence similarity; strain 52 was most closely related to Stenotrophomonas rhizophila with 98.55% sequence similarity; strain 6 was most closely related to Stenotrophomonas rhizophila with 96.50% sequence similarity，which might represent a novel species of the genus． The phenotypic study showed that: strains 6，11 and 52 were Gram negative，straight rodshaped，did not produce extracellular lipase and amylase，possessed strong proteolytic activity．The optimal temperature and pH for the enzyme activity were 55℃ and 6. 7 for the protease from strain 6，40℃ and 8. 5 for the protease from strain 11 and 65℃ and 7.4 for the protease from strain 52.［Conclusion］The research firstly introduced the distribution of Stenotrophomonas and Chryseobacterium specieses in the Arctic marine water，extended the diversity of the proteaseproducing bacteria from the Arctic，and provided a useful basis for further study of cold-adapted protease．

Abstract:Abstract:［Objective］Genetic diversity of the rhizobial bacteria nodulating Tibetia himalaica in the eastern part of Qinghai-Tibet Plateau (Ganzi State，Sichuan) were evaluated．［Methods］The pure culture method was used for isolating the rhiobial strains from the nodules． BOXAIR，16S rDNA sequences，16S rDNA PCR-RFLP and Principal Component Analysis (PCA) were used to determine the genetic diversity and phylogenetic relationships．The growth in different salt contents，pH and temperatures were tested．［Results］In total 22 strains were isolated from 12 samples in 8 counties．The strains tested were classified into 4 clusters in 16S rDNA PCR-RFLP，and 8 clusters were formed in BOXAIR． The 16S rDNA Simpson genetic diversity index was D=0.872. The strains tested were closely related to Rhizobium (11/22 strains)，Mesorhizobium (4 /22 strains) and Rhizobium-Agrobacterium (7 /22 strains)．All strains could regularly grow in YMA medium amended with 1 % NaCl，and 15/22 strains could grow in 4 % NaCl． SCAU679，SCAU694and SCAU706 could adapt to 7% NaCl，while SCAU689 could tolerant 8% NaCl．Among the strains tested，15 strains could grow in the pH range from 4.0 to 11; 16 isolates grew well from 4 to 45℃，and all of 22 strains could grow at 28 ℃ after treatment at 60℃ for 10 min．［Conclusion］This study revealed the high genetic diversity of the rhizobia isolated from Tibetia himalaica in the eastern part of Qinghai-Tibet Plateau． The strains tested were adapted to high salt，different range of temperatures and pH．

Abstract:Abstract:［Objective］In 2012，the cumulative mortality of farmed sturgeons in Beijing was almost 60% with various symptoms，including the reddening of the anus with yellow exudation，ascities in the peritoneal cavity，petechial haemorrhages in liver and internal muscle wall，and the swollen spleen．［Methods］We isolated the pathogen from the dying sturgeons with significant pathological signs，and then analyzed its morphological，physiological and biochemical characteristics，taxonomic status，and drug sensitivity． Moreover，the pathogenic characteristic of presumptive pathogens was identified by artificial infection．［Results］The 16S rDNA sequence of the pathogen was more than 99% homology with that of Plesiomonas shigelloides，suggesting that the pathogen was P．shigelloides，which was also demonstrated by the results of biochemical tests．The LD50 of the pathogen to sturgeon was 1.0×105.8 CFU/mL，and it also can cause liver，kidey and spleen to lesions. There were no activities of amylase，caseinase，lipase，gelatinase and haemolysis of extracellular products of P. shigelloides，and its toxicity might be from endotoxin．In addition，the bacterium was specific sensitive to enrofloxacin，doxycyline hyclate，florfenicol and thiamphenicol with MIC less than 2μg/mL．［Conclusion］P. shigelloides was the main pathogen to cultured sturgeons in Beijing area，and enrofloxacin，doxycyline hyclate and florfenicol can be used against the disease.

Abstract:Abstract:［Objecive］To identify the interaction between Newcastle disease virus (NDV) matrix (M) protein and avian nucleophosmin B23.1 in HEK-293T cells．［Methods］Specific primers used to amplify M gene and B23.1 gene were designed and synthesized according to JS/5/05/Go whole gene sequence (JN631747) and avian nucleophosmin B23.1 gene sequence (NM_205267)．Viral RNA and cellular RNA were extracted from allantoic fluid of NDV JS/5/05/Go strain and DF1 cells with TRIzol reagent，respectively．The M gene and B23. 1 gene were amplified by RT-PCR and then subcloned into eukaryotic expression vectors to generate the recombinant plasmids pEGFP-M，pCMV-HA-M and pDsRed-B23.1．To investigate the localization features of M protein and B23. 1 protein，we transfected the plasmids pEGFP-M and pDsRed-B23. 1simultaneously into HEK-293T cells and observed the results by fluorescence microscopy．We further confirmed the interaction between the two proteins by co-immunoprecipitation (Co-IP) assays． ［Results］ The fusion proteins were successfully expressed in transfected HEK-293T cells by Western blot analysis．The NDV M protein and avian nucleophosmin B23. 1 showed co-localization features in the nucleolus in co-transfected HEK-293T cells．Furthermore，the binding of M with B23.1 was demonstrated by Co-IP assays．［Conclusion］These results suggested that NDV M protein interacted with avian nucleophosmin B23.1 and the nucleolar localization of M might be regulated via interaction with B23.1.

Abstract:Abstract:［Objective］We studied the regulating effect of human bocavirus 1 (HBoV1) nonstructural protein NP1 on the activity of cellular transcription factors and the expression of inflammatory cytokine TNF-αand IL-6．［Methods］ The modulation of NP1 was measured by the Dual Luciferase Reporter Assay System and the expression of cytokines TNF-αand IL-6 was detected by ELISA and Real-time PCR． The luciferase based mammalian two-hybrid system was used to analyze whether the function of NP1 protein aroused from oligomerization． ［Results］The transcription factors AP-1，STAT3 and STAT1 but not NF-κB were up-regulated by NP1，which was evidenced by approximately 2-3-fold increase of the luciferase activity compared to the control vector．Moreover，NP1 increased the TNF-α mRNA expression，but not contributed to cytokine IL-6 secretion． We also found that the self-interaction did not exist when NP1 was solely expressed in 293T cells．［Conclusion］This study demonstrates for the first time that NP1 may play important roles in activation of transcription factors and up-regulation of inflammatory cytokines expression，suggesting that NP1 be involved in HBoV1 pathogenesis．

Abstract:Abstract:［Objective］To evaluate the influence of DNA extraction methods on the actinobacteria diversity analysis in saline environment via 16S rDNA Restriction Fragment Length Polymorphism．［Methods］CTAB-SDS method，glass bead beating method and repeated freezing and thawing method were used to extract total DNA in soil samples from the Yanqi Salten． The 16S rDNA clone libraries were constructed by using the purified 16S rDNA PCR amplicons to transform the E．coli DH5α．The transformants in the library were further analyzed by RFLP．The unique 16S rDNA clones were sequenced and further used for phylogenetic analysis．［Results］Different Operational Taxonomic Units (OTU) were obtained from DNA extracts and total 35 OTUs were obtained from CTAB-SDS method，19 OTUs from galss bead beating method and 14 OTUs from repeated freezing and thawing methods．Up to 52% OTUs in the three libraries constructed displayed lower similarity with the published sequence，perhaps representing novel taxons．The total OTUs belong to Actinobacteridae，Acidimicrobidae and Rubrobacteridae subclasses．［Conclusion］ DNA extraction methods influence the actinobacterial diversity． Each of the DNA extraction method in our study has some drawbacks and biases，so it is better to use combined DNA extracts from different DNA methods to evaluate the microbial diversity in salty environments.

Abstract:Abstract:Kelimycin，is a new macrolide antibiotic drug obtained through genetic engineering approaches．With 4 "-Oisovalerylspiramycins as the major components，was produced by genetically engineered Streptomyces spiramyceticus transformed with 4 " -O-acyltransferase gene from S． mycarofaciens．［Objective］ Improve the efficiency of strain fermentation，to meet the needs of industrial production．［Methods］ The enhanced kelimycin-producing strain was obtained by applying various conventional mutagenesis approaches，and high-throughput screen methods，including protoplast mutagenesis by ultraviolet，mutagenesis by diethyl sulfate and UV-reactivation，valine content resistance screen and enrichment of improved strains．A strategy for positive mutant enrichment was developed after mutagenesis and before high-throughput screen．［Results］ Finally，the high-producing strain WSJ-1-7-49-133-82-18-43 was obtained and its potency in shake flask increased by 56% compared to the original strain． The potency in 500 L pilot fermenter increased by 61%．［Conclusion］This study shows that the screening industrial production strains can be enhanced effectively by combining multiple conventional mutagenesis and high-throughput screen methods.

Abstract:Abstract:［Objective］Resistance of Listeria monocytogenes was determined through its survival rate and intracellular pH (pHi) after exposed to in vitro digestion models.［Methods］Simulated gastrointestinal systems were prepared according to the major natural components in saliva，gastric juice and contents of the small intestine．Survival of L．monocytogenes was estimated by plate counting and pHi measured via fluorescence ratio imaging microscopy.［Results］L．monocytogenes had a survival rate of over 90% after saliva treatment. Viability was low (less than 0.05%) in gastric juice with pH at or below 3.0 and in the subsequent intestinal fluid. The survival rate started to increase in gastric juice at pH 3.5 and was higher between 11.2%－85.9% for the two tested strains at pH 4.0．The pHi was similar between saliva-treated and untreated bacteria as revealed by fluorescence ratio imaging microscopy. The pHi remained relatively stable at or higher than 7.75 in gastric juice at pH 3.5 and 4.0 or with subsequent introduction of intestinal fluid.［Conclusion］ L．monocytogenes could tolerate gastric juice with pH at or above 3.5 as shown by its good survivability and stable pHi，most probably by maintaining integrity of its membranes.