Abstract:Abstract: Marine viruses play great roles in the marine ecological system such as modulating the biodiversity and species population, regulating the nutrient cycling, intervening gene transfer and influencing climate changes. Recent research achievements on marine viruses were reviewed in this paper. We focused on the modulating role of marine viruses in marine ecosystem and discussed future research perspectives.

Abstract:Abstract: The genus Sphingomonas was established in 1990. Sphingomonas spp. synthesize sphingans, structurally related biopolymers such as gellan, welan and diutan. At present, only gellan is applied widely in foods and pharmaceuticals. The economic value of other sphingans has not been well explored, and related research of sphingans still remains limited. In the present review, we address the latest taxonomy developments of Sphingomonas, details about structure, characteristics and biosynthetic pathway of sphingans, current knowledge on the molecular genetics and genetic engineering of sphingans. In addition, we indicate future research needs.

Abstract:Abstract: Spiroplasma spp. are helical, motile bacteria that lack cell wall and flagellum, and are enclosed within a single membrane with their genomes ranging from approximately 0.78-2.20 Mb in size, the smallest among known self-replicating prokaryotes. So they have been used as model organisms for studying movement, metabolisms and sex ratio. Currently, 34 serological groups are recognized; three of these groups encompass 15 subgroups of inter-related strains. To date, 37 species among all serogroups and subgroups have been given binomial names. Complete characterization of a new species involves numerous phenotypic and genotypic tests as outlined in the minimal standards document, including phylogenetic data and a reevaluated set of required phenotypic and genotypic tests. Spiroplasma spp. are most often found in association with insects and plants flowers, and the interactions of Spiroplasma/host can be classified as commensal, pathogenic or mutualistic. Investigation of spiroplasma resources in China and research on their biodiversity will undoubtedly improve our understanding of these important microbial resources.

Abstract:Abstract: [Objective] In order to study the diversity of endophytic endospore-forming bacteria in Cinnamomum longepaniculatum. [Methods] We took modified nutrient agar medium for isolation and cultivation and analyzed the 16S rRNA gene sequences of the isolates. [Results] Forty non-redundant endospore-forming bacterial isolates were ascertained, which accounted for 38.1% of all the endophytic bacterial isolates. Of them, 24 isolates were from roots, 7 from stems and 9 from leaves. Phylogenetic analysis based on 16S rRNA gene sequences showed that 35 of them belonged to 16 species of the genera Bacillus, Lysinibacillus and Paenibacillus, and 5 isolates with <97% sequence similarities to their closely related members were presumed to be potential novel species. [Conclusion] The results showed that the cultivable endospore-forming bacteria diversity was abundant and there were some potential nove1 strains in Cinnamomum longepaniculatum. The microflora of endophytic endospore-forming bacteria in individuals of C. longepaniculatum showed that some bacteria distributed in different organs, but the others were organ-specific bacteria.

Abstract:Abstract: [Objective] We studied the role of the nitrogen fixation gene PST1305 located within the nitrogen fixation island of Pseudomonas stutzeri A1501. [Methods] We constructed the mutant strain (np1305) by homologous recombination and triparental conjugation, and determined the nitrogenase activity by the acetylene reduction test. Through RT-PCR, we analyzed the transcriptional units of PST1305 gene and its nearby genes. Real-Time PCR was applied to compare the expression level of PST1305 gene between optimal and non-nitrogen fixating conditions. [Results] Compared to the wild type, the nitrogenase activity in mutant strain (np1305) was partially decreased, however, functional complementary strain (np1305Comp) could restore nitrogenase activity close to wild type level. PST1305 gene was co-transcribed with its upstream gene (nifB and fdxN) and downstream gene (nifQ, PST1303 and PST1302). In contrast to the nitrogen excess conditions, expression of PST1305 under nitrogen-fixing conditions was significantly upregulated for 38.7-fold. [Conclusion] Disruption of PST1305 exhibited a declined nitrogenase activity compared to the wild type A1501. PST1305 gene might participate in biological nitrogen fixation by involving in the electron transport or the oxygen protection mechanism of nitrogenase. These results suggested that PST1305 gene was a new gene required for optimal nitrogenase activity of Pseudomonas stutzeri A1501.

Abstract:Abstract: [0bjective] To express Luciferase gene in Escherchia coli through developed Deinococcal bacteria - E. coli shuttle expression vector. [Methods]The D. bacteria - E. coli shuttle expression vector pZT17 was constructed based on plasmids of pUE30, pGBM5 and pKatCAT. Then pZT17 with lux+ from Photinus pyralis was used to transform into D. grandis and E.coli. The recombinant strains were induced separately. [Results] Based on a small cryptic plasmid from Deinococcus radiopugnans, a shuttle vector between Escherichia coli and deinococcal bacteria was constructed. The plasmid vector could stably aintained in Deinococcus grandis under non-selective conditions. Moreover, it is showed that a luciferase gene was highly expressed both observed in D. grandis and E.coli. [Conclusions]The D. bacteria - E. coli shuttle vector was constructed successfully, the developed shuttle vector makes it possible to induce expression of DNA damage and repair gene from Deinococcus species.

Abstract:Abstract: [Objective] We reconstructed two recombinant plasmids and studied their effects on L-threonine accumulation of Escherichia coli W3110. [Methods] We amplified the threonine operon containing ThrLp promoter, lead peptide thrL, thrA thrB and thrC genes by PCR from E. coli W3110 chromosome and ligated it into the pMD19 T-vector. Site-directed mutation were carried out by gene splicing by overlap extension PCR to release the feedback inhibition of aspartokinase I (thrA). Two recombinant plasmids WYE112 and WYE134 were transformed into E. coli W3110 by electroporation. Fed-batch cultures of E. coli W3110 were carried out in 5-Liter fermentors and the L-threonine concentration was measured by HPLC. [Results] Fed-batch fermentation results showed that E. coli W3110 could accumulate little L-threonine (0.036 ± 0.004 g/L) but recombinant E. coli W3110 harboring the plasmid WYE112 containing a threonine operon exhibited a L-threonine production of 2.590 ± 0.115 g/L. Furthermore, L-threonine production reached 9.223 ± 1.279 g/L when the feedback inhibition of thrA was released. [Conclusion] Overexpression of threonine operon can lead to the accumulation of L-threonine. Further release of feedback inhibition of aspartokinase I can enhance its accumulation.

Abstract:Abstract: [Objective] β-glucosidase can be used to prepare gentiooligosaccharide from glucose. The purpose of this study is to obtain β-glucosidase through DNA recombinant technology as well as to optimize the production of gentiooligosaccharide by the recombinant β-glucosidase. [Methods] We cloned bgl, the gene encoding β-glucosidase from Aspergillus niger (CMI CC 324626) into the expression vector pPIC9K to construct the recombinant plasmid pPIC9K- bgl. The vector was then transformed into Pichia pastoris KM71 for extracellular overproduction of β-glucosidase. The activity of the expressed enzyme was measured by the assay of transglucosidation reaction and the transglucosidation product was identified by HPLC and LC-MS. Furthermore, the condition for prepare gentiooligosaccharide by this recombinant β-glucosidase is optimized.[Results] A. niger β-glucosidase was successfully expressed in P. pastoris and the recombinant produced gentiooligosaccharide from glucose. In addition, the main operation parameters of this enzymatic conversion were optimized. At 80% glucose, 60℃, pH 4.5, 1 mmol/L K+, 60 U β-glucosidase per gram substrate, and 48 h reaction time, the gentiooligosaccharide produced reached 50 g/L. [Conclusion] This is the first report of producing gentiooligosaccharide by recombinant β-glucosidase.

Abstract:Abstract: [Objective]: To study the impact of GSH (glutathione) on the gene expression of exoY and exoS in Pseudomonas aeruginosa. [Methods]: We treated P. aeruginosa with BSO (buthionine sulfoximine) and DEM (diethylmaleate) to deplete GSH, or construct the P. aeruginosa mutant containing a lacZGm disrupted gshB (glutathione synthetase) gene by homologous recombination technology. The expression of exoY and exoS was determined by measuring light production of the lux-based reporters on pMS402. [Results]: The expression of exoY and exoS decreased in the gshB mutant and P. aeruginosa treated with BSO and DEM. [Conclusion]: GSH in the P. aeruginosa can increase the expression of the genes exoY and exoS. Furthermore, this result provided possibilities to elucidate the molecular mechanisms of pathogenesis and immune response triggered by P. aeruginosa.

Abstract:Abstract: [Objects] To purify and characterize bacteriocin produced by L. paracasei HD1.7. [Methods] Paracin 1.7 was purified by chromatography and its molecular weight was measured by using sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE). Antibacterial activity was measured by using the agar-well diffusion method. [Results] The bacterial strain for the fermentation was identified as Lactobacillus subsp. paracasei. Paracin 1.7 had the activity of inhibiting the growth of other bacteria. Maximum production of Paracin 1.7 was in the stationary phase. Paracin 1.7 can be well purified with Cation exchange chromatography, Sephedex (G50, G25, G10) gel chromatography and high performance liquid chromatography (HPLC) on C18 column and its determined molecular weight was about 11kDa by Tricine-SDS-PAGE. Paracin 1.7 shows a broad spectrum of activities against various strains in the genera of Proteus, Bacillus, Enterobacter, Staphylococcus, Escherichia, Lactobacillus, Microccus, Pseudomonas, Salmonella and Saccharomyces, some of which belong to food borne pathogenic bacteria. Although Paracin 1.7 displayed stability toward heat and acidic pH, it was sensitive to several proteolytic enzymes. The inhibitory activities remain well after stored at 4oC for 4 months. [Conclusion] Paracin 1.7 can be a potential food preservative on the basis of its antibacterial characters.

Abstract:Abstract: [Objective] We analyzed bacterial colonization associated with spores of arbuscular mycorrhizal fungi (AMF) Gigaspora margarita, to indicate their ecological niche, and to provide information for further researches on their populations or functions. [Methods] Six bacteria strains (Peanibacillus sp. M060106-1, Peanibacillus sp. M061122-2, Peanibacillus sp. M061122-6, Bacillus sp. M061122-4, Bacillus sp. M061122-10 and Brevibacillus sp. M061122-12) isolated from G. margarita spores were tagged with green fluorescence protein (GFP) using the carrier plasmid pNF8 (gfp-mut1). We analyzed the ecological niche and population dynamics of tagged strains on G. margarita under different conditions by using fluorescent microscope and/or plate counts. [Results] Four strains (M060106-1, M061122-6, M061122-10 and M061122-12) were tagged with GFP, showing high plasmid stability. These tagged strains possessed the basic characteristics identical to their original strains and, hence, were fit for short-term study of environmental colonization. All four GFP-tagged strains colonized the spore wall of G. margarita, and M061122-6 and M061122-12 further colonized the fungal hyphae. Under different pH conditions, the population dynamic of each GFP-tagged strain on the spores showed the same trend, i.e. first increased and then decreased, and the effects on the population size varied with different pH value. GFP-tagged strains colonized the spores of low viability more easily than those of high viability, and the population dynamic on the spores of high viability was different for each tagged strain. [Conclusion] The isolated bacteria associated with G. margarita spores can re-colonize the fungal spores, whereas their colonizing ability depends on their characteristics and environmental factors. These data contributes to the further understanding of populations and functions of AMF-associated bacteria.

Abstract:Abstract:【Objective】We screened dominating microbial species isolated from aging flue-cured tobacco and studied their aroma improving effect.【Methods】Total DNA of microorganisms from the fermentation flue-cured tobacco surface of NC89, ZhongYan 100 and ZhongYan 101 were extracted. Under the PCR-DGGE, the diversity of microorganisms on fermentation tobacco leaves were studied and dominating microbial species were screened. We further studied the influence of dominating microbial species on the content of aroma components of the fermentation flue-cured tobacco.【Results】1) By using DGGE analysis, there were 5 dominant bands A, B, C, D and E in all tobacco leaves samples of the three varieties; In further studies, five dominant DGGE bands were isolated, cloned and sequenced. From them we screened a dominant microorganism. 2) The content of most aroma components in tobacco leaves increased when they were sprayed with the dominant microorganism, comparing with the control.【Conclusion】The dominant microorganism can improve the flavor of tobacco leaves during ripening.

Abstract:Abstract:【Objective】To clarify pathogens and sources of the off-plate syndrome at the attachment stage in the larval culture of Apostichopus japonicus, and further to find out effective medicines for this disease.【Methods】 Etiological analysis was performed on larvae with typical off-plate syndrome from three larvae culture factories. Suspicious pathogens were used for artificial infection test, and were identified through morphological, physiological and biochemical tests, and 16S rDNA sequence analysis. Quantitative bacterial analysis was done on the culture systems of the three factories, including water sources, rearing water, ordure (in the pond floor), attachments and feeds. Finally, drug-sensitive tests were done against the pathogens.【Results】A common dominant bacterium strain was isolated from all ill larvae included in the study. Artificial infection test showed it was the causative pathogen associated with the disease, and the artificially infected sea cucumbers had same syndromes to the naturally ill ones. The bacterium was identified as Vibrio sp. Bacterial quantity of water sources was in the qualified range (<50 cfu/mL), while out of the standard range in others (>1 × 105 cfu/mL). The sources of the pathogen were complicated, since pathogens were discovered in the water sources, rearing water, ordure, attachments and feeds. However, the density of causative bacteria was the highest in the feeds, middle in the attachments, and lowest in the water sources. Twelve antibiotics could inhibit growth of the pathogens.【Conclusion】The possible pathogen for off-plate syndrome was Vibrio sp. Feeds may be the main source of the pathogen. Twelve antibiotics besides nalidixic acid could be applied for disease prevention and treatment of Apostichopus japonicus.

Abstract:Abstract: [Objective] The mrkD gene encodes the adhesin which mediates Klebsiella pneumoniae to adhere human respiratory tissue.We aimed to analyze the adhesion mechanism and adhesion block funcntion of MrkD adhesin. [Methods] The recombinant glutathione-S-transferase(GST)-tagged adhesive protein(MrkD) was expressed in E. coli and was purified to homogeneity using GST affinity chromatography. The GST tag was cut by thrombin to obtain the MrkD protein that was identified by SDS-PAGE and Western blot. The adhesive activity of MrkD was examined by adhesive experiments and the binding site was observed by laser confocal microscopy. [Results] The adherent activity of Klebsiella pneumoniae was significantly inhibited by the MrkD. These experimental data demonstrated that the MrkD inhibited the adhesion of Klebsiella pneumoniae. [Conclusion] Our results suggest that MrkD adhesin contains the adhesion epitopes.The future work will be carried out to identify the epitopes and characterize them, then to optimize the combination presentation of these epitopes to develop an efficient vaccine for Klebsiella pneumoniae.

Abstract:Abstract: [Objective] To prepare a vaccine with inactivated Feline Panleukopenia Virus (FPV) isolated from Siberian tigers and to evaluate its immunological effect. [Methods] FPV-HLJ, an FPV strain previously isolated from Siberian tiger in our lab was used to inoculate cat kidney cell line F81 with dose 1/10(v/v) using the synchronizing inoculation method. Inoculated F81 cell line was cultured at 37°C and collected when cytopathic effect was up to 75%. Viral suspension was inactivated by using formaldehyde for 24 h and inactive vaccine preapred by adding aluminium hydroxide gel as adjuvant to the suspension. The inactive vaccine was applied to 2- month old kitten tigers of hypodermically after protection effect was proved in 2-month old nonimmunized cats by using the same vaccination procedure. [Results] Antibody level in vaccinated cats revealed an increasing trend that the valence of antibody reached 1:1024~2048 after three times of vaccination. All vaccinated cats survived challenges of virulent FPV virus. The valence of antibody reached 1:1024 in most vaccinated tigers 15 days after the third vaccination. [Conclusion] The results indicated that the inactivated vaccine can produce immunoprotection for tigers.

Abstract:Abstract: [Objective] To study and characterize secretive expression of canine parvovirus capsid protein 2 (VP2) gene in eukaryotic cells. [Methods] To construct secreting expression vector of VP2 gene, we obtained CD5 signal peptide (SP) DNA fragment from plasmid containing human CD5 SP DNA sequence and inserted the fragment into multiple clone site of eukaryotic expression vector pcDNA3.1A. The canine parvovirus VP2 gene was amplified by PCR and inserted into expression vector pcDNA3.1-CD5sp down stream of CD5 SP. The recombinant pcDNA-CD5sp-VP2 plasmids were transfected into HEK293T cells mediated by calcium phosphate. VP2 binding activity for canine transferrin receptor was analyzed by ELISA method. [Results] Recombinant pcDNA-CD5sp-VP2 plasmid proved to be correct by sequencing. VP2 proteins were detected by Western-blot in the culture medium of transfected 293T cells, which indicated that the expressed VP2 protein could be secreted into the medium mediated by human CD5 SP. VP2 protein had the activity to bind canine transferrin receptor(TfR). [Conclusion] The secreting expression of VP2 in eukaryotic cells was achieved by using human CD5 SP. Recombinant VP2 showed the ability to bind canine TfR.

Abstract:Abstract: [Objective] To evaluate the immunity of the adenovirus recombinant rAd-HA-GFP encoding an H3N2 swine influenza virus hemagglutinin. [Methods] Thee groups of 6-week-old pigs (5 pigs per group) were vaccinated intramuscularly with the recombinant, one group was immunized with 10-8 TCID50 recombinant adenovirus rAd-HA-GFP, the other groups were vaccinated with 2×10-8 TCID50 and 4×10-8 TCID50. A control groups (5 pigs) were vaccinated with recombinant adenovirus rAd-GFP. virus-specific hemagglutination-inhibition(HI) antibody was detected by 2-6 weeks post vaccination. Compared the difference of vaccinated intramuscularly, intragastric administration and intranasal inoculation，thee groups of different HI antibody pigs and one control group were challenged with a virulent H3N2 field virus. [Results] The results showed that pigs in the groups given higher immunizing dose were developed higher levels HI antibody. All of vaccinated intramuscularly, intragastric administration and intranasal inoculation could produce HI antibody, but the titer of vaccinated intramuscularly was higher significantly(P<0.01).Three groups of pigs(5 pigs per group) which HI titers were 1:80，1:160，1:320 respectively were challenged with a virulent H3N2 field virus, include the negative control group. The immunization efficacy was evaluated by clinical signs,the rate of virus isolation and HI titer. It suggested that immunological response induced by rAd-HA-GFP could resist attack of SIV effectively. [Conclusion] The adenovirus vaccines rAd-HA-GFP are efficacious for SIV and have the additional advantage over commercial vaccines that suckling piglets have no pre-existing maternally-derived antibody to block early life vaccination.

Abstract:Abstract: [Objective] Shiga-like toxin-producing Escherichia coli (STEC) causes edema disease in piglets and hemolytic uremic syndrome in human. Shiga-like toxins (Stxs) produced by STEC induce mammalian cells death via either necrosis or apoptosis. However, the ability of stx2e, separated from edema disease (Stx2e), to trigger apoptosis and the sequence of intracellular signaling events have not yet been completely defined. In this study we investigated the apoptotic effects of Stx2e on Vero cells. [Methods] Vero cells were treated with different concentrations of Stx2e for different time and the apoptotic cells were characterized by acridine orange and ethidium bromide fluorescent dye staining. The fragmentation of chromatin from Vero cells treated with Stx2e were detected by agarose gel electrophoresis. The expression patterns of apoptosis-associated factors were assayed by Western blotting. [Results] Stx2e-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies, whereas ricin did not induce apoptosis of Vero cells even at a high dose. Fluorescent dye staining showed that Stx2e induced apoptosis of Vero cells in dose- and time-dependent manners. Caspase-3 was activated whereas expression levels of bcl2 associated X protein (Bax) and caspase-9 had no change compared with the negative control. [Conclusion] Stx2e induced intensively apoptosis of Vero cells, which was mediated through the mitochondrion-independent pathway and might be receptor-dependent pathway.

Abstract:Abstract: [Objective] To enhance bacteriocin production by Lactobacillus paracasei HD1.7 immobilized with calcium alginate gel by statistical methods. [Methods] A 26?2 two-level fractional factorial design was employed first where 6 variables were studied for their influence on bacteriocin production. The concentration of sodium alginate, inoculation volume and culture time were the most significant variables to improve bacteriocin production. The greatest response area was estimated by the design of steepest ascent approach. A three-level Box–Behnken factorial design was employed for maximizing the bacteriocin production and the number of cell cycle. The repeated batch fermentation using immobilized cells was done. [Results] The optimal condition for bacteriocin production and the number of cell cycle was with sodium alginate 2.8%, CaCl2 5%, immibilization time 4 h, the density of entrapped cells 1/20, culture time 63 h and inoculation volume 8.2%. The period of fermentation was 24 h of a batch fermentation after 63 h and bacteriocin production was 70% of free-cell fermentation. [Conclusion] After optimizing, the total production of bacteriocin was increased by 19% using the batch fermentation during 327 hours, the beads were intact with little cell leakage and repeatedly used, which laid foundation for the high study of bacteriocin and finally extracted.

Abstract:Abstract: [Objective] The fastidious property of human adenovirus type 41 (Ad41) may be resulted from inadequate expression of protein V (pV), the minor core protein of adenovirus, in packaging cells. In this report, we prepared antiserum to pV of Ad41 and studied the mechanism of Ad41 fastidiousness. [Methods] Coding sequence of pV was amplified by PCR with the genome DNA of wild Ad41 (NIVD103) as template, and cloned into pET30a(+) vector to generate a recombinant plasmid called pET-pV. His-tag-fused pV was expressed in pET-pV-transformed E. Coli strain BL21(DE3) by adding the inducer of Isopropy β-D-1-Thiogalactopyranoside (IPTG) and purified with the method of immobilized metal ion affinity chromatography (IMAC). Antiserums to pV were collected from pV inclusion bodies-immunized mice and evaluated by Western blot. [Results] The sequencing assay showed that the cloned pV gene was highly homologous with that of Ad41 Tak strain, and there were only three residues changed in the corresponding amino-acid sequence. pV was expressed as inclusion bodies or in soluble form in BL21(DE3) cells under inducing condition of 1 m mol/L IPTG, 37 °C, 4 h or 0.5 m mol/L IPTG, 25 °C, 8 h, respectively. Antiserums to pV from most immunized mice were highly effective for Western blot assay. After infected with equivalent Ad41, 293E12, an Ad41 E1B55K-transfected 293 cell line, expressed more pV than 293 cells. [Conclusion] We successfully prepared antiserums to Ad41 pV and it could be used in Western blot assay to study the fastidious property of Ad41.

Abstract:Abstract: [Objective]: In order to construct the recombinant bovine hepervirus-1(BHV-1) which expressed foot and mouth disease virus (FMDV)VP1 gene, we constructed a BHV-1 gE gene transfer vector by inserting the synthetic VP1 gene of FMDV (O/China/99) under the immediate-early promoter of cytomegalovirus. [Methods]: The mixtures of parental virus (BHV-1/gE-/LacZ+ ) DNA and transfer vector was transfected into bovine turbinate cells using calcium phosphate-mediated transfection. Then the propagated viruses were harvested. The recombinant BHV-1 (designated BHV-1/gE-/VP1) was obtained by selection for white virus plaques. [Results]: PCR results showed that VP1 gene was successfully inserted into the genome of BHV-1/gE-. The expression of VP1 in infected cells was proved by indirect immunofluorescence assay and Western blotting. [Conclusion]: The research provided a basis for development of BHV-1 vector vaccines for FMD and other important bovine infectious diseases.