Abstract:Abstract:Because the integrase of class 1 integron in bacteria has been demonstrated closely relative with antimicrobial resistance，we summarize in this review the discussions on some regulation factors with expression of the integrase，such as antibiotics，carbon catabolite repression (cAMP-CＲP complex)，the promoter of gene cassette variants (Pc-P2 combinations)，attC sites，some nucleotide-associated proteins，and the length of variable region． Furthermore，we also provide knowledge of the regulation mechanism of integrase，as well as insight into controlling the dissemination of antimicrobial resistance in the environments．

Abstract:Abstract:Bacterial resistance is a threat to public health． Bacterial biofilm formation is one of the main reasons for persistent infection caused by bacteria． Biofilm development is a complex process that involves many factors and genes which play various roles in all stages of the biofilm formation． This review focuses on the gene regulatory mechanisms relate to the biofilm formation of Staphylococcus，the most common pathogen that causes nosocomial infection，as well as the latest developments of pharmacological anti-biofilm therapies． We also address new strategy to treat bacterial infection and the development of drugs and vaccines against biofilm resistance.

Abstract:Abstract:Klebsiella pneumoniae is of great attractiveness because it naturally produces a series of bulk chemicals such as 1，3-propanediol，2，3-butanediol and 3-hydroxypropionic acid．Although this species has been fueled in recent years，its pathogenicity is considered an obstacle hindering industrial applications． Here we portray a picture of the virulence factors，including pili，receptors，capsular polysaccharides，lipopolysaccharides，and newly identified virulence factors．This review covers aspects of virulence genes，proteins，metabolic activities，as well as the mechanisms underlying infection and immune responses． Based on state-of-the-art advances in metabolic engineering and synthetic biology，the strategies for eliminating or attenuating the virulence of K． pneumoniae were proposed，and the feasibilities for these protocols were also briefly discussed.

Abstract:Abstract:［Objective］This study aimed to identify soft rot pathogens of Chinese cabbage ［Brassica campestris L. ssp．chinensis (L.) Makino var． communis Tsen et Lee］in Beijing.［Methods］The 40 strains isolated from Tongzhou and Daxing districts in Beijing were characterized by morphological，biological，biochemical and physiological methods，16S rRNA sequence as well as 16S-23S rRNA intergenic spacer (IGS) region analysis.［Results］The strains belonged to two different Pectobacterium carotovorum subspecies: 13 strains of them belonged to Pectobacterium carotovorum subsp．carotovorum (Pcc) and the other 27 strains belonged to Pectobacterium carotovorum subsp． brasiliensis (Pcb). The results of Chinese cabbage (Brassica campestris L. ssp．pekinensis) pathogenicity test showed that the strains in the same subspecies，origins and 16S rRNA gene sequences had significant differences in pathogenicity.［Conclusion］Pectobacterium carotovorum subsp． carotovorum and Pectobacterium carotovorum subsp．brasiliensis were the soft rot pathogens on Chinese cabbage ［Brassica campestris L. ssp．chinensis(L.)Makino var． communis Tsen et Lee］in Beijing．It was the first report that Pectobacterium carotovorum subsp． brasiliensis (Pcb) caused soft rot disease on cabbage in China．

Abstract:Abstract:［Objective］Type III secretion system (T3SS) is essential for many phytopathogenic bacteria to cause disease in susceptible host plants and to elicit a hypersensitive response in resistant host and non-host plants．Xanthomonas campestris pv． campestris (Xcc) uses T3SS to deliver T3SS effectors (T3SEs) directly into host cells，where they play important roles in pathogenesis．The aim of this study was to identify a new T3SE in Xcc．［Methods］ To validate if XC3176 is a T3S effector translocated into plant cells，the promoter and signal region of XC3176 were fused to the plasmid pLJB of harboring HＲ-inducing AvrBs1 C-terminal domain lack of 58 N-terminal amino acid residues． The recombinant plasmid pLJB3176 was introduced by triparental conjugation into ΔavrBs1 and ΔhrcV． Hypersensitive response induced by the obtained strains ΔavrBs1/pLJB3176 and ΔhrcV/pLJB3176 were examined on the pepper ECW-10R． To determine transcription of XC3176，GUS fusion report strains were constructed． The virulence of Xcc strains was investigated on the Chinese radish by the leaf-clipping method．［Results］Hypersensitive response was elicited on the pepper ECW-10R by the strain ΔavrBs1/pLJB3176，but not ΔhrcV/pLJB3176．The GUS activities in the mutant strains ΔhrpX and ΔhrpG were significantly lower than that in the wild type Xcc strain．The mutant of XC3176 reduced virulence significantly and the complementary strain C3176 could restore the virulence as the wild-type strain．［Conclusion］XC3176 is a T3SSdependent effector of Xanthomonas campestris pv． campestris． The expression of XC3176 is regulated by hrpG and hrpX．XC3176 is required for the full virulence of Xcc 8004.

Abstract:Abstract:［Objective］To further study physiological functions and structure of β-glycosidase，we cloned the bglC gene of Bacillus subtilis and expressed it in E．coli BL21(DE3)，followed by the characterization and structural simulation of the enzyme.［Methods］We amplified the bglC gene and transferred it into E. coli BL21(DE3)，then we obtained a mutant with higher hydrolytic activity by directed evolution．After purifying the enzymes through a nickel-nitrilotriacetic acid agarose column，we characterized the wild-type and mutant enzymes．By means of CD spectrum，Native-PAGE and protein 3-D structure modeling，we analyzed the higher structure of the β-glycosidase.［Results］We got one mutant enzyme BSGLY_M1 (A242T/T385A/S425L) with improved hydrolytic activity by directed evolution and screening． The specific activity of wild-type enzyme was 9. 7 U/mg，with optimum temperature at 60 ℃ and optimum pH at 7.0．The specific activity of BS-GLY_M1 was 17.1U/mg，with optimum temperature at 55 ℃ and optimum pH at 7.0．Moreover，the halflife time of the mutant enzyme at 55 ℃ was 3.5 h，2 h longer than that of wild-type enzyme． Furthermore，the catalytic efficiency (Km/Kcat) of BS-GLY_M1 on the substrates 4-nitrophenyl-β-galactoside，lactose，and arbutin improved obviously．The polymer forms of the enzyme under the native conditions were of dimer and tetramer，but the dimer was the most probable functional unit．Result of structural simulation also showed slight changes occurred in the tertiary structure of the mutant enzyme，which may be the main reason for the enhanced thermal stability and catalytic efficiency of BS-GLY_M1.［Conclusion］β-glycosidase from Bacillus subtilis could be expressed in E.coli BL21 (DE3)，meanwhile its hydrolysis efficiency could be further improved by directed evolution.

Abstract:Abstract:［Objective］To clone the NADPH gene (PuNOX) and Glyoxal oxidase gene (PuGLOX) from a medicinal fungus Polyporus umbellatus，and to carry out the bioinformatic analysis．［Methods］We used the Rapid Amplification of cDNA ends (RACE) technique to obtain the full length cDNA of these two genes． We used a series of bioinformatic tools to characterize physiochemical properties of the two deduced protein．The analyses of multiple alignment and phylogenetic trees were performed using Bioeditor and MEGA 5. 0 softwares．［Results］The entire cDNA of PuNOX and PuGLOX were 1674 bp，1723 bp in length and encoded a 557-amino acid protein and 515-amino acid protein with a molecular weight of 63.845 kDa and 55. 891 kDa and the isoelectric point of 5. 58 and 4. 82，respectively． PuNOX had high identities (74 to 80%) with NADPH peroxidase from other fungus． From the evolutionary tree，PuNOX was closely related to that of Pleurotus ostreatus． PuGLOX had high identities (＞50%) with Glyoxal oxidases from various fungus． Phylogenetic tree analysis suggested that PuGLOX was closely related to that of Phanerochaete chrysosporium.［Conclusion］ Molecular characterization of the two oxidative stress related genes will be useful for further functional determination of the genes involved in the sclerotium development of Polyporus umbellatus．

Abstract:Abstract:［Objective］ In order to detect the effect of lactic acid bacteria isolated from Tibetan Plateau on silage fermentation quality of Elms nutans．［Methods］ We used 3 isolated lactic acid bacteria with better growth at low temperatures of 10 and 15 ℃ at ensiling of Elymus nutans． Subsequently，effects of the selected lactic acid bacteria on fermentation profiles of Elymus nutans silages stored at 15 and 25 ℃ were evaluated by using the same species of commercial inoculants as the control．［Results］PP-6 isolated from Tibetan Plateau could ferment raffinose，lactose，sorbitol，melibiose and sucrose，and LS-5 could ferment cottonseed sugar，laetrile，rhamnose，lactose，sorbitol，xylose，arabinose，melibiose and sucrose，but the same species of commercial strains could not use these sugars． Inoculation of these three strains into Elymus nutans at 15 and 25 ℃ ensiled for 50 d，we found that LS-5 significantly reduced silage pH，propionic acid concentration and ratio of ammonia nitrogen/total nitrogen at 15 ℃ (P＜0.05)，salvaged more water-soluble carbohydrate and crude protein; Application of LP-2 and PP-6 as a combined inoculant to Elymus nutans significantly improved lactic acid concentration (P＜0.05)，resulting in a lower ratio of ammonia nitrogen/total nitrogen，saved more crude protein and significantly reduced neutral detergent fiber content (P＜0. 05) as compared with the commercial strains．［Conclusion］The three isolated strains can improve silage quality of Elymus nutans growing on the Qinghai-Tibetan Plateau at low temperature，but these strains have no obvious advantages at 25 ℃ in comparison with the commercial inoculants.

Abstract:Abstract:［Objective］We studied the conditions of culturing Bacillus subtilis SY1 to treat cotton stalk alkaline peroxide mechanical pulping waste water，and evaluated afterwards the safety of its release to plants and animals.［Methods］The culture conditions were optimized through single factor tests，including temperature，initial pH，oxygen，and inoculation size． We used rats and big ear rabbits to test the safety of treated waste water.［Results］The optimal condition was as follows: initial pH7.0，hydraulic retention time 30 h，temperature 30 ℃，filling rate of aeration 16 L/h，the dosage of Bacillus subtilis SY1 0.8 g a liter water，and the stuffing volume accounting for 30% of the effective volume of the reactor．Under these culture conditions，the live spore number of Bacillus subtilis SY1 reached 6.27×109 CFU/mL．The removal rate of COD reached 70.4%．Results of the acute dermal toxicity test，dermal irritation test，eye irritation test and skin sensitization test after treated by the fermentation showed no clinical signs or changes conditions，and no deaths during the test period of 21 days． Animal body weight had a tendency to increase and had no abnormalities in the major organs，the Lethal Dose 50 (Abbreviated LD50) values was equal or greater than 10.5 g/(kg body weight) in acute dermal toxicity experiments．LD50 values were equal or greater than 2500 mg/(kg body weight) in dermal irritation experiments． No rabbits exhibited chemosis eye at any time during the test period． The results of skin sensitization test showed no erythema or edema． The skin sensitization value was less than 0.5.［Conclusion］ Pulping waste water of APMP cotton stalk fermented by Bacillus subtilis SY1 had reduced COD and no obvious toxicity to animals.

Abstract:Abstract:［Objective］ Displaying cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans 251 on the cell surface of Saccharomyces cerevisiae to improve 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) production.［Methods］CGTase encoding gene cgt was inserted into the 3' terminal of Aga2p of vector pYD1 and the obtained recombinant plasmid pYD1-cgt was then transformed into S.cerevisiae EBY100 to produce surface displayed CGTase and culture conditions (culture medium，inductive temperature and concentration of inducer galactose) were optimized． Moreover，resulted CGTase displayed on the yeast cell surface was used for the AA-2G biosynthesis under the optimized condition.［Results］ CGTase activity on the cell surface of recombinant yeast，S.cerevisiae EBY100-pYD1-cgt，reached 0.5 U/ml in 48 h fermentation using Yeast Peptone Galactose culture medium with 20% galactose as sole carbon source and inducer at 25 ℃．The displayed CGTase exhibited better thermostability and pH stability than that of free CGTase．The concentration of AA-2G produced by the surface displayed CGTase was 37% higher than that produced by free CGTase at its optimal transformation conditions of 30 ℃ and pH4.5．［Conclusion］The cell surface display system based on a-agglutinin is an effective system for displaying CGTase． During AA-2G production by surface displayed CGTase，the by-product glucose might be consumed by yeast cell and thus facilitated AA-2G production． The whole cell EBY100-pYD1-cgt will have better prospects for applications．

Abstract:Abstract:［Objective］ Bdellovibrio-and-like organisms (BALOs) are small-sized，parasitic bacteria that rely on other Gram-negative bacteria for survival． Our work aimed to characterize the community diversity of BALOs associated with tropical shrimp ponds．［Methods］We collected water samples from eight shrimp ponds culturing Litopenaeus vannamei，in Zhanjiang，China，and extracted total DNA of the samples．Then the 16S rＲNA gene clone libraries were constructed following PCR amplification with BALOs family-specific primers． The community composition and structure of BALOs were further analyzed based on phylogenetic analysis．［Results］A total of 726 and 664 valid clones were obtained from the 16S rＲNA gene libraries of families Halobacteriovoracaceae and Peredibacteraceae，respectively． Subsequently，they were respectively grouped into 68 and 44 OTUs (operational taxonomic units) at 99.5% sequence similarity，and assigned into 37 and 28 clusters at 97% similarity． For the Halobacteriovoracaceae libraries，most clusters were so far uncultured with the exception of 5 members，which accounting for 43.5% clones． And the cultivable cluster IX and the uncultured cluster B28 were the first and second dominant ones respectively． For the Peredibacteraceae libraries，the cluster pa12 and the only cultivable cluster A3.12 were the first and second dominant ones respectively．Notably，the values of Shannon diversity indices of Halobacteriovoracaceae and Peredibacteraceae tend to be reversed with the change of salinity，but the total BALOs diversities among the eight shrimp ponds were similar.［Conclusion］Shrimp mariculture ponds in Zhanjinag harbor a high diversity of BALOs comprising of Halobacteriovoracaceae and Peredibacteraceae，and salinity affect their community structure and composition greatly.

Abstract:Abstract:［Objective］For exploring and characterizing the diversity of virus-like particles from salt mines in Yunnan，China.［Methods］ Virus-like particles were identified from enriched brine or saline soil samples by transmission electronic microscopy and double-layer plate，and their morphological properties were further characterized.［Results］Three types of virus-like particles，head-tailed virus，linear virus and spherical virus，were observed by using transmission electronic microscopy． Then，two Halomonas viruses and one Chromohalobacter virus were subsequently isolated from Qiaohou salt mine and Yipinglang salt mine． According to the sample sources，their morphological characteristics and the character of host strains，those viruses were named as Qiaohou Halomonas Siphoviridae Virus 1(QHHSV-1)，Yipinglang Halomonas Siphoviridae Virus 1 (YPHSV-1) and Yipinglang Chromohalobacter Pleomorphic Virus 1 (YPCPV-1)．QHHSV-1 was head-tailed virus with a diameter of 47 nm icosahedrons-like head，and an easily broken 75 nm length tail．YPHSV-1 was head-tailed virus with a diameter of 50 nm icosahedrons-like head，and a 140 nm length tail．YPCPV-1 was a pleomorphic，variable-sized (20－50 nm) virus，which possessed protuberances on the virus particles.

Abstract:Abstract:［Objective］To optimize the cultural conditions including adhesive materials and salinity for one tropical benthic diatom (Amphora sp． HN08)．［Methods］Two experiments were performed: (1) five adhesive materials including agar，glass，PVC plate，plastic film and nylon net were used to culture Amphora sp． HN08 HN08; (2) Amphora sp． HN08 was cultured under 6 salinity levels from 1 through 6%．The algal cells were harvested after 9 days treatments，and the biomass productivity，adhesive strength and pigments content of cells were examined.［Results］ For the adhesive materials experiment，cultures with glass and plastic plate showed the highest biomass production and the dry weight of cells reached to 3.64 g/m2．The adhesion strength level of cells on the glass plate was III degree，which means the cells were easy to be separated from the glass and dewatered by centrifugation． Salinity did not contribute significantly to the biomass production，while it significantly influenced the cells adhesion and pigments concentration． The adhesive strength levels of the cells under 3% or 4% salinity was IV degree，which led to an easy harvest process． While the cells cultured under high salinity (5% and 6% ) usually suspended in the medium and were difficult to be dewatered by centrifugation． Both chlorophyll a and carotenoid content of cells cultured with higher salinity (≥3%) are extremely higher (p＜0.01) than that with lower salinity (1% and 2%)．Chlorophyll a and carotenoid content of cells under 5% salinity was 26. 27% and 11. 11% by dry weight，respectively．［Conclusion］Taking the harvest and biomass production together，we think the optimal salinity for Amphora sp． HN08 was between 3% and 4%，and the glass plate was suitable for adhesive material．However，considering the cost and safety，the PVC plate is suggested to be used for biomass production．

Abstract:Abstract:［Objective］ To compare the antigenicity，adhesion inhibition activity and immune protection of the serum immunized with recombinant adhesins Clumping factor A (ClfA)，Fibronection binding protein A-A (FnBPA-A) and Fibronection binding protein A-BCD (FnBPA-BCD) of Staphylococcus aureus.［Methods］ClfA，FnBPA-A and FnBPABCD genes were expressed and the resulting recombinant proteins were induced and purified．After immunizing mice with these purified proteins in combination or alone，the serum antibodies of mice were analyzed and compared for their antigenicity，adhesion inhibition activity and immune protection.［Results］ The recombinant FnBPA-BCD protein had better fibronection (Fn) binding ability than that of fibrinogen (Fg) binding and ClfA． FnBPA-A had better binding capacity to Fg than that of FnBPA-BCD．The antibody titer induced by ClfA and FnBPA-A were better than that of FnBPA-BCD，and three proteins co-immunization group had better antibody titer and adhesion inhibition to Staphylococcus aureus than those of single recombinant protein immunization group (P＜0.05)．The immune protection rate for both ClfA and three proteins co-immunization group were 100% and for FnBPA-A and FnBPA-BCD immunization group the immune protection rate were 80% and 50%，respectively．［Conclusion］ Our data suggest that coimmunization with these 3 recombinant proteins helps to achieve better immune effect.

Abstract:Abstract:［Objective］To analyze NLRC4 inflammasome responses in macrophages induced by C-terminal of Escherichia coli EscI protein． ［Methods］ NLRC4 inflammasome responses in mouse peritoneal macrophages were analyzed after delivery of the peptides containing C-terminal amino acid sequences of E.coli EscI protein in vitro.［Results］ The peptides containing C-terminal 15 amino acids of EscI protein could significantly activate NLRC4 inflammasome responses in macrophages pre-stimulated with lipopolysaccharide． Intracellular caspase-1 was activated and pyroptotic dead cells were found after peptides delivery． The contents of cytokines，IL-1β and IL-18，in supernatants were elevated significantly compared with that of the control (P＜0.05)．Besides，through comparison of IL-1β contents under different stimulation conditions，4 h incubation after peptides delivery (peptides: lipofectamine 2000=70 μg/μL) could obviously promote the secretion of IL-1β． ［Conclusion］ Peptides containing C-terminal 15 amino acids of E.coli EscI protein can significantly induce NLRC4 inflammasome activation in macrophages.

Abstract:Abstract:［Objective］ We studied the phenotypic characterization of Phytophthora parasitica Dastur var．nicotianae．［Methods］ Phenotypic characterization of the pathogen was studied to provide information for disease management program by using BIOLOG phenotype MicroArray (PM)． Using PM plates 1 to 10，950 different phenotypic characterizations were tested.［Results］ P．parasitica was able to metabolize 74% of tested carbon sources，96% of nitrogen sources，100% of sulfur sources，and 98% of phosphorus sources． Most informative utilization patterns for carbon sources of P． parasitica were organic acids and carbohydrates，and for nitrogen were various amino acids． The pathogen presented 285 different nitrogen pathways． It had wide range adaptabilities in osmolytes with up to 1% sodium chloride，up to 3% potassium chloride，up to 5% sodium sulfate，up to 20% ethylene glycol，up to 2% sodium formate，up to 5% urea，and up to 2% sodium lactate． It also exhibited active metabolism under pH values between 3.5 and 10，with optimal pH of around 7.0．The pathogen showed both decarboxylase and deaminase activities in the presence of various amino acids．［Conclusion］These phenotypic characterizations of P．parasitica provided the theoretical basis for the next study of the pathogen in physiology and metabolism，and provided potential new way for tobacco black shank management.