Abstract:Abstract: Copper is an essential trace element in all organisms and serves as a catalytic cofactor for many biological processes in cells. Yet excess cuprous and cupric forms can be high toxic to the cells. Thus cells must have developed diverse mechanisms to control the uptake and distribution of copper. Much are known about the copper metabolism in Saccharomyces cerevisiae and a few other fungi. In this review, we focus on the recent research in copper uptake, transport and distribution in model organism baker’s yeast Saccharomyces cerevisiae, as well as the new frontier in other fungi, e.g. the novel roles of copper in the pathogenesis of the fungal pathogen Cryptococcus neoformans.

Abstract:Abstract: Vibrio parahaemolyticus is a halophilic gram-negative bacterium that causes food borne acute gastroenteritis in human being or certain diseases in aquatic species. In addition to thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh), two sets of type III secretion systems (T3SS) were recently found to be associated closely with virulence. T3SS1 located on chromosome 1 is involved in cytotoxicity to host cells and orchestrates a multifaceted host cell infection by induction of autophagy, cell rounding, and eventual cell lysis. T3SS2 in chromosome 2 is enterotoxic. In this we address the composition of the two T3SS, their functions and regulation in Vibrio parahaemolyticus.

Abstract:Abstract: Quorum sensing (QS) is a phenomenon that microbes regulate some of their genes by signals related to the density of population. It is confirmed that acyl-homoserine lactones (AHL), some peptides, some furanones and some other small moleculars can be used as quorum-sensing signals by microbes. Microbes control their physiology with different QS systems in parallel or hierarchical ways. A lot of microbial pathogenesis connect with quorum sensing closely. More and more studies show that QS systems regulate in microbial pathogenesis through the following points: (1) QS helping pathogens invasion and colonization; (2) QS regulating production of virulent factor; (3) QS giving pathogens the ability of immunity or drug resistance. We review the role of QS in microbial pathogenesis and address a new way to prevent and control microbial diseases.

Abstract:Abstract: [Objective] Based on the molecular diversity information, seven actinomycete-selective culture media and isolation conditions were modified to isolate and cultivate diverse rare actinomycetes from Hymeniacidon perlevis. [Methods] Modified, selective cultivation and enrichment media were used, with the addition of an elemental solution of simulating the elemental composition of marine sponge H. perlevis. Restriction Fragment Length Polymorphism (RFLP) analysis of 16S rDNA sequence was used to reveal the diversity of culturable rare actinomycetes. [Results] A total of 59 actinomycete strains were isolated from the marine sponge H. perlevis. A total of 27 representative actinomycetes were selected according to their morphological feature, color and pigments. They gave 15 different RFLP patterns after digesting their PCR products of 16s rDNA with Hha I. The results showed that these isolates belonged to 10 genera: Streptomyces, Nocardiopsis, Micromonospora, Cellulosimicrobium, Gordonia, Nocardia, Prauseria, Pseudonocardia, Saccharomonospora and Microbacterium. [Conclusion] The modified isolation media and selective cultivation procedures are highly effective in the recovery of culturable actinomycetes from the marine sponge H. perlevis, resulting in the highest diversity of culturable rare actinomycetes from any sponges.

Abstract:Abstract: [Objective] Plant rhizosphere bacteria play an important role in biogeochemical cycles. Microbial diversity of cultivable bacteria from soil-plant-mineral system in a mine tailing was assessed. [Methods] Cultivable bacteria were isolated by plating and screening from plant root, rhizosphere and bulk soils of predominant plants in a mine tailing of Nanjing, Jiangsu Province. Phylogenetic analyse based on 16S rDNA sequence comparisons and amplified rDNA restriction analysis of isolates were investigated. [Results] In total, 60 pure cultures were isolated; they could be grouped into 18 different operational taxonomic units (OTU) at the similarity level of 60%. Nineteen bacterial strains belonged to eleven genera (Rhizobium, Pseudomonas, Pantoea, Arthrobacter, Microbacterium, Bacillus, Paenibacillus, Acinetobacter, Sphingomonas, Kocuria, Mitsuaria) of three major phylogenetic groups (Proteobacteria, Actinobacteria, Firmicutes). Rhizobium, Pseudomonas and Pantoea were the dominant groups. [Conclusion] Different cultivable bacteria inhabited in roots and soils of dominant plants in the mine tailing. They might play a certain role in the soil-plant-mineral environment.

Abstract:Abstract:[Objective] OxyRxoo is a homologue of OxyR from Xanthomonas oryzae pv. oryzae (Xoo),the pathogen of bacterial blight of rice. To elucidate the role of OxyRxoo in detoxification of hydrogen peroxide.[Methods] OxyRxoo was studied by gene cloning, sequencing, deletion, complement and phenotype analysis. [Result]Compared to those of the wild-type strain PXO99A, there was no difference in bacterial growth in vitro of △oxyRxoo, but △oxyRxoo is more sensitive to H2O2 with reduced catalase activity. In the presence of H2O2, the expression of catalase genes (ahpCxoo, catBxoo, katExoo and srpAxoo) was significantly down-regulated, while the expression of oxyRxoo was up-regulated in △oxyRxoo. [Conclusion] OxyRxoo functions as a transcription regulator in mediating and controlling H2O2 detoxification in Xoo.

Abstract:Abstract: [Objective] A drawback of the expression of single chain antibody fragment (scFv) in prokaryotic system is the protein accumulation in the cytoplasm as inclusion body. We aimed at high-level production of an anti-aflatoxin B1 scFv in functional form. [Methods] The gene of scFv-H4 was cloned into pET22b vector and transformed into E.coli BL21(DE3) and Origami (DE3), respectively. The amount of functional scFv-H4 was optimized in terms of IPTG concentration and induction temperature. [Results] scFv-H4 could be expressed in both BL21(DE3) and Origami (DE3). Compared with BL21(DE3), Origami(DE3) could express multifunctional scFv-H4 (35 mg/ml) and less in inclusion body (11% of the total expression). The expression of scFv-H4 was significantly affected by induction temperature rather than IPTG concentration. [Conclusion] The pET22b could be used for high-level expression of the functional scFv-H4 in Origami (DE3), which has an oxidative cytoplasm. In addition, the induction at low temperature avoided the formation of inclusion body.

Abstract:Abstract: [Objective] To identify and analyze bioactive compounds of an actinomycete strain Z-L-22 suppressing Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato. [Methods] Morphological, biological and biochemical characterization, chemotaxonomy analysis and 16S rDNA sequences homology analysis were performed to identify the strain Z-L-22. Bioactive compounds were separated and retrieved by thin layer chromatography. Paper chromatography and confirmation tests were used to identify the antibiotic. PCR was carried out using the primers targeted to synthetase of the antibiotic. [Results] Strain Z-L-22 belonged to Streptomyces sp. and was similar to Streptomyces setonii. Two main bioactive components were isolated by thin layer chromatography, which were all identified as actinomycin. New actinomycin synthetase gene was cloned using the primers designed from actinomycin synthetase conserve domain. [Conclusion] Strain Z-L-22 was classified as Streptomyces setonii. Actinomycin produced by Streptomyces setonii was first reported.

Abstract:Abstract: [Objective] This study was carried out to isolate and characterize an agarase from a marine bacterium Agarivorans albus QM38. [Methods] SDS-PAGE grade agarase was obtained from the fermentation broth after removing the bacteria by centrifugation, ammonium sulfate precipitation, DEAE- sepharose fast flow anion exchange chromatography and Sephacryl S-100 gel filtration. Enzyme’s molecular weight was determined with SDS-PAGE. The catalysates of the isolated enzyme were determined with mass spectrography. [Results] Agarase A was isolated. The molecular weight of agarase A was 127.80 kDa. More characterizations of agarase A were studied and the results showed that the optimal reaction condition for agarase A was at 35 ℃, pH 7.6, and agar concentration of 0.9% (w/v), while most of the metal ions inhibited the activity of it. The catalysates of agarase A were mainly tetrose and hexose. [Conclusion] Agarase A was purified from the medium. It could hydrolyze jellied agar and yield simple catalysates. Its molecular weight is different from all the agarases reported so far.

Abstract:Abstracts: [Objective] In order to identify the predominant strains of polycyclic aromatic hydrocarbon (PAH)-degrading consortia harboring in sea water and surface sediment collected from deep sea of the Middle Atlantic Ridge. [Methods] We employed enrichment method and spread-plate method to isolate cultivable bacteria and PAHs degraders from deep sea samples. Phylogenetic analysis was conducted by 16S rRNA gene sequencing of the bacteria. Then we analyzed the dominant bacteria in the PAHs-degrading consortia by denaturing gradient gel electrophoresis (DGGE) combined with DNA sequencing. [Results] Altogether 16 cultivable bacteria were obtained, including one PAHs degrader Novosphingobium sp. 4D. Phylogenetic analysis showed that strains closely related to Alcanivorax dieselolei NO1A (5/16) and Tistrella mobilis TISTR 1108T (5/16) constituted two biggest groups among the cultivable bacteria. DGGE analysis showed that strain 4L (also 4M and 4N, Alcanivorax dieselolei NO1A, 99.21%), 4D (Novosphingobium pentaromativorans US6-1T, 97.07%) and 4B (also 4E, 4H and 4K, Tistrella mobilis TISTR 1108T, ＞99%) dominated the consortium MC2D. While in consortium MC3CO, the predominant strains were strain 5C (also 5H, Alcanivorax dieselolei NO1A, ＞99%), uncultivable strain represented by band 5-8 (Novosphingobium aromaticivorans DSM 12444T, 99.41%), 5J (Tistrella mobilis TISTR 1108T, 99.52%) and 5F (also 5G, Thalassospira lucentensis DSM 14000T, ＜97%). [Conclusion] We found that strains of genus Alcanivorax, Novosphingobium, Tistrella and Thalassospira were predominant bacteria of PAHs-degrading consortia in sea water and surface sediment of Middle Atlantic Ridge deep sea, with Novosphingobium spp. as their main PAHs degraders.

Abstract:Abstract: [Objective] Our research objective is to obtain the active substances from culture liquid of Hypoxylon perforatum with inhibitory effect on Sphaeropsis sapinea growth and germination. [Methods] Water and ester were used for extracting active substances from culture liquid of Hypoxylon perforatum, either by extracting directly from cultural liquid, or extracting under ultrasonic. Growth-inhibiting rate and germination-inhibiting rate were used as the index of antifungal activity. [Results] Five extracts from culture liquid of Hypoxylon perforatum showed antifungal activities to Sphaeropsis sapinea growth and germination. [Conclusion] Ethyl acetate–extract from culture liquid of Hypoxylon perforatum has a highly antifungal activity to Sphaeropsis sapinea, high stability in the natural environment. So it has a highly development of the value and application prospect. The mainly inhibiting active substance is methyl p-methoxy cinnamate, which as medicine intermediate was used in cosmetic to be UV-protection absorbefacient. Diisobutyl phthalate and Dibutyl phthalate are all the common plastic-enhancer for PolyvinylChloride (PVC). It is very important in medicinal industry and chemistry industry to discovering the natural presence of the three compounds.

Abstract:Abstract: [Objective] To isolate and identify a new ionizing-radiation resistant strain capable of surviving under highly ionizing radiation conditions as UV and gamol/La radiation and to characterize its radioresistant properties. [Methods] The isolates were sampled from Radiation Centre of Guangxi University, Nanning, China, and the medium used for isolation and cultivation of the bacterium was General Bacterial Medium (GBM). The new ionizing-radiation resistant strain WGR700T was identified by its morphology, biochemical and physiological characteristics, fatty acids, G+C content of DNA, UV and gamol/La radiation resistance and 16S rRNA gene sequence homology. [Results] The strain WGR700T is of rod-shape, Gram-negative, non- spore-forming, non motile, aerobic and red-pigmented. The optimum temperature and pH for strain WGR700T growth is 37°C and pH7.0, respectively. The predominant respiratory quinone is MK-8 and its cell well contains ornithine. The major cellular fatty acids found in the cell wall are 16:1ω7c, 16:0, 15:1ω6c, iso-15:0 and iso-17:0. DNA of strain WGR700T had a G+C content of 64.7mol%. WGR700T was highly resistant to UV (>728 J/m2) and gamol/La radiation (D10 = 9.8 kGy). Phylogenetic analysis of the 16S rRNA gene sequences showed 87.1-95.6% similarities with other recognized Deinococcus species.[Conclusion] Based on the high 16S rRNA gene sequence divergence and phenotypic differences, it is proposed that the new isolated strain should be classified as a novel member in the genus Deinococcus with the name Deinococcus guangxiensis sp. nov. The type strain is WGR700T (= CGMCC 1.7045T = CICC 10360T = JCM 15082T ).

Abstract:Abstract: [Objective] To obtain carbendazim-degrading microbial strains, and to use them for bioremediation of contaminated soil. [Methods] A carbendazim-degrading bacterium T2-2 was isolated from the screening of drug-tolerated mutants Trichoderma strains. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis showed the presence of the metabolites after shake incubation of the Trichoderma T2-2 at temperature 25℃, 200r/min in mineral salt medium that contained 100mg/L carbendazim. We prepared T2-2 bioremediation agents from crop straw through solid fermentation. By inoculating T2-2 in soil, we performed a bioremediation test of sterilized soil and original soil at 0.1mg/g dry soil of carbendazim concentration and 107cfu/g dry soil of inoculating amount. In addition, we also conducted a control effect experiment of T2-2 against fusarium wilt of cucumber. [Results] The metabolites detected by HPLC-MS were 2-aminobenzimidazole, benzimidazole, and 2-aminobenxinitrile in the culture filtrate after 2 days of incubation. Carbendazim and metabolites could no longer be detected through the High-pressure liquid chromatography (HPLC) analysis in the culture filtrate after 5 days of incubation. In the soil bioremediation test, carbendazim in the sterilized soil was degraded completely after 6 days of inoculation, whereas the process only needed 4 days in original soil. It showed crop straw could function as co-metabolic substrate and promote co-metabolism of T2-2 and indigenous microorganisms. Moreover, the efficiency of T2-2 against cucumber fusarium wilt might reach 81.7%, which is superior to chemical pesticide. [Conclusion] T2-2 could degrade carbendazim in soil and thus control plant disease.

Abstract:Abstrat: [Objective] Ayu (Plecoglossus altivelis) vibriosis threatens ayu aquaculture seriously caused by mass mortality due to severe infections. We characterized the vibriosis pathogen of ayu in Ninghai country. [Methods] A dominant strain was isolated and identified by a series of biochemical and physiological tests. The lethal dose 50% (LD50) was calculated by the modified Karber’s method. PCR amplification and sequence analysis were used to further identify the pathogen. [Results] LD50 of ayu-H080701 was 1.2×104 CFU to ayu. PCR amplification showed that the bacterial universal primers for 16S rRNA gene and the specific primers for the metalloprotease (MP) gene of Listonella anguillarum worked. 16S rRNA gene analysis showed that ayu-H080701 shared 99.4%-99.5 % nucleotide identical to L. anguillarum isolates, while 94.3% and 91.9% nucleotide identical to L. pelagius and Photobacterium damselae respectively. MP analysis showed that ayu-H080701 shared 97.6%-98.8 % amino acid sequence identical to L. anguillarum isolates, while lower than 75.6 % to other bacteria. Phylogenetic analysis showed that ayu-H080701 grouped constantly with L. anguillarum isolates. [Conclusion] The biochemical, physiological tests and sequence analysis all strongly supported the identification of the pathogen causing ayu vibriosis in Ninghai country, China, as an isolate of L. anguillarum.

Abstract:Abstract: [Objective] To explore a new therapeutic strategy against acute hepatitis B and fulminant hepatitis B, we studied effect of co-immunization with HBV DNA and HBsAg on the T cell proliferation reaction. [Methods] We immunized the BALB/c mice with HBV DNA vaccine (pcDS2) plus HBsAg by intramuscular injection. The immunization was performed on week 0, 2 and 4. The anti-HBs（IgG）antibody titer, T lymphocyte proliferation reaction , and the expression of IL-10 and Foxp3 in CD3+ T cell were detected on week 6. [Results] The anti-HBs IgG titer induced by pcDS2 plus HBsAg group was higher than that induced by pcDS2, or HBsAg alone. Compared to mice immunized with pcDS2, or HBsAg alone, the stimulated index (SI) of T cell proliferation induced by the pcDS2 plus HBsAg group tested by MTT methods decreased. Besides, the immune suppression of T cell proliferation response induced by co-immunization group was further confirmed by flow cytometry. Finally, the expression of IL-10 and Foxp3 in CD3+ T cell was up-regulated in the co-immunization group significantly. [Conclusion] The co-immunization of HBV DNA vaccine and HBsAg can induce the humoral immune response, but cannot induce antigen specific T cell proliferation reaction. Besides, the immune suppression induced by co-immunization may be correlated with the expression of IL-10 and Foxp3.

Abstract:Abstract: [Objective] To construct an infectious full-length cDNA clone of Asia1/JS/China/2005 strain with Arg-Gly-Asp (RGD) receptor recognition site. [Methods] We constructed foot-and-mouth disease virus type Asia1 full-length cDNA clone pFMDV-RGD by using site-directed mutagenesis. The plasmid pFMDV-RGD contained the desired mutation. The plasmids pFMDV-RGD were linearized with NotI enzyme. Linearized plasmid and pcDNAT7P plasmids expressing T7 RNA polymerase cotransfected into BHK-21 cells to rescue FMDV-RGD. [Results] We constructed FMDV Asia1/JS/China/2005 strain full-length cDNA clone with Arg-Gly-Asp receptor recognition site by sequence. We obtained rescued virus by plasmid cotransfection. The results of sequencing, indirect immunofluorescence, electron microscope and sulk mice pathogenicity analysis showed foot-and-mouth disease virus containing Arg-Gly-Asp receptor recognition site was successfully rescued. [Conclusion] The results lay a foundation for further study of biology characteristic diversity of rescued virus with Arg-Gly-Asp and Arg-Asp-Asp (RDD) receptor recognition site.

Abstract:Abstract: [Objective] To establish a reverse genetic system of rabies virus for producing safe and efficient rabies vaccine. [Method] By reverse genetic and molecular cloning technique, we established two rabies virus rescue systems including (1) help plasmids expressing N, P and L protein, and (2) CMV/T7 and T7 promoter. [Results] Wild-type rERA-VC was rescued by both systems, and had the same growth kinetics as parental virus. The third generation virus could grow to high titer. [Conclusion] The established reverse genetic system for rescuing wild-type rERA-VC provides the possibility of producing safe and efficient rabies virus vaccines.

Abstract:Abstract: [Objective] To understand mutations of avian influenza virus (AIV) under the antibody selective pressures. [Methods] We continuously passed an H9N2-AIV strain LG1 in embryos with or without maternal antibody to LG1 in 6 separate serials. At the 10th, 20th, 30th, 40th and 50th passages in each serial, H9 hemagglutinatin gene (HA) sequences were determined and compared to the original LG1 strain. [Results] Only unstable random mutations happened in 29 sites with nonsynonymous vs synonymous (N/S) mutation ratio of 1.42 during 50 passages in 2 serials without maternal antibodies. However, multiple stable nonsynonymous mutations were detected gradually during 50 passages of 4 serials in embryos with maternal antibody to LG1 strain, and the NS/S ratio was as high as 3.46 among 45 mutated sites. [Conclusion] The results suggested that the antibody selective pressure influenced mutations of H9N2 AIV HA gene during the passages in embryos with maternal antibody. Embryos with maternal antibody to certain viruses could be used as experimental models to study the immune selective pressures on viral mutations.

Abstract:Abstract: [Objective] To better understand the host inflammatory responses, in particular inflammatory cytokines responses of Vesicular Stomatitis virus (VSV) infection, and the host-VSV interaction. [Methods] We used VSV Indian strain to infect the Intestinal Pig Epithelial Cell Jejenum (IPEC-J2) cell. Then we measured and analyzed the viral RNA by using real-time PCR. The transcript level of cytokines (IL-2, 6, 8, 10,12, IFN-α, IFN –γ, TNF-α, TGF-β and TLR3) were detected by real-time PCR. [Results] We found that the transcript levels of IL-6, IL-8, IL-12, TNF-α and TLR3 were increased obviously whereas TGF-β showed no significant difference. TPEC-J2 cell did not secrete IL-2, IL-10, IFN-α and IFN–γ. [Conclusion] The level of inflammatory response was increased when VSV infected TPEC-J2 cell.

Abstract:Abstract：[Objective] The purpose of this research is to establish a simple rapid amplification of cDNA ends (RACE) strategy for direct mapping of the 3’ end and 5’ end of the genomic RNA of Newcastle disease virus (NDV), and to analyze the leader and trailer sequence of NDV strains belonging to different genotypes. [Methods] Classic RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) was specifically modified for mapping both ends of the NDV genome. 3’-RACE was carried out by genomic RNA ligation with 5’ end phosphated adaptor CL+, and the 5’ end was obtained by first strand cDNA with adaptor CL+. [Results] A modified RLM-RACE strategy was established in this paper, which proved simple, low-cost, repetitive and could be specifically used to map genome ends of NDV. By using this method, the leader and trailer sequence of 5 NDV strains, termed JS/5/05/Go, JS/07/04/Pi, JS/07/16/Pi, JS/7/05/Ch and JS/9/05/Go, belonging to genotype III, VI and VII was determined, respectively. [Conclusion] The initial 8nt at the 3’ and 5’ ends of the genome of genotype I-VI NDV strains were complementary, whereas, the complementary sequences of strain JS/5/05/Go were up to 9 nt due to a mutation from T to C at the 9th nt in the 5’ end. The 3’ end of NDV genomic and anti-genomic RNA was predicted to form a potential hairpin structure. The U→C（T→C）mutation was located in the circle part of the hairpin in the 5’ end of anti-genomic RNA, and had no visible influence on the formation of RNA secondary structure. However, the sequence of the circle part of the hairpin was changed from 3’-UUUC-5’ to 3’-UCUC-5’, more similar to the 3’-UCUUA-5’ in the hairpin of genomic RNA.

Abstract:Abstract: [Objective] To clone promoter-active fragments from Corynebacterium glutamicum for further construction of expression vectors. [Methods] Random Sau3A I digested fragments of C. glutamicum 10147 chromosome were shot-gun cloned into the promoter-probe vector pAKC6 and promoter activity of the inserted fragments was selected by chloramphenicol resistance of transformed C. glutamicum cells. [Results] Thirty promoter-carrying fragments were isolated. Three C. glutamicum clones harboring pAKC6 with promoter fragments displayed chloramphenicol acetyltransferase (CAT) activity of more than 24 U/mg. The fragment F57 led to the highest CAT activity of 32.50 U/mg, even more than that produced by the promoter Ptrc, 26.33 U/mg. [Conclusion] The strength of promoter on fragments F21, F54 and F57 is as strong as promoter Ptrc in C. glutamicum. These fragments can be used to construct expression vector.