Abstract:Abstract: The overview of projects funded by general programs, key programs and national science fund for distinguished young scholars in discipline of microbiology, National Natural Science Foundation of China (NSFC) from 2006 to 2010 was recommended. Some important characters such as the distribution of projects in different subjects, organizations, regions and research fields were analyzed. Some important research fields which should be supported in “The Twelfth Five-Year Plan” was also put forward. The goal of the paper is to provide information of funding by NSFC for researchers in microbiology fields.

Abstract:Abstract: It is gradually recognized that host specificity and pathogenicity of influenza A virus could also be affected by proteins other than hemagglutinin (HA) due to its polygenic nature. In this review, detailed description was focused mainly on neuraminidase (NA), polymerase basic protein 2 (PB2) and nonstructural protein 1 (NS1), especially their molecular determinants associated with virulence and host range restriction of influenza A virus.

Abstract:Abstract: Wine microbes can produce volatile sulfur compounds during wine fermentation. The main volatile sulfur compounds found in wine are hydrogen sulfide, sulfides, thiol, thiolesters, sulful-containing fusel alcohols, sulful-containing heterocycles and “fruity volatile thiols”. They can affect the wine flavor dramatically. Metabolism pathways and related gene regulations of these compounds are discussed in this review. This review also proposes that research on wine microbes is an effective way to increase concentrations of flavor compounds in wine and inhibit undesirable off-flavor compounds at the same time.

Abstract:Abstract: Amylopullulanse (E.C. 3.2.1.1/41) has the enzymatic activities of both α-amylase (E.C. 3.2.1.1) and pullulanase (E.C. 3.2.1.41), and is classified into the glycoside hydrolase 13 and 57 families. Amylopullulanse can hydrolyze both the α-1,4 and α-1,6 glucosidic bonds and is valuable for decreasing cost, increasing both efficiency and dextrose equivalent in starch processing industry. Thermostable amylopullulanase is more valuable in starch saccharification industry due to its capability to catalyze both the liquefaction and saccharification processes under the industrial starch liquefaction condition. In addition, the special bifunctional catalytic mechanism of amylopullulanase is also of great value in enzymology research. The present review focuses on the structure and function of amylopullulanases and provides a brief overview on the latest studies.

Abstract:Abstract: [Objective]To better reveal the functions of key members involved in cyclic di-GMP signal metabolism pathways in the rice bacterial blight pathogen rice Xanthomonas oryzae pv. oryzae (Xoo). [Methods] vieAxoo (PXO_04753), a gene putatively encoding the EAL domain proteins was investigated by gene deletion mutation using the marker exchange, complementation and phenotypic analysis. [Result] The sequence of vieAxoo cloned from genomic DNA of the wild-type strain PXO99A was found to be highly conserved in plant-pathogenic Xanthomonas spp. VieAxoo was structurally featured with EAL and REC domains. No significant changes in virulence to rice, EPS production and flagellar motility were found in △vieAxoo compared to PXO99A, whereas remarkable changes in induction of hypersensitive responses (HR) in tobacco and biofilm formation were observed. [Conclusion] VieAxoo might function as an important regulator in cyclic di-GMP signaling and regulation of bacterial induction of HR and biofilm formation of Xoo.

Abstract:Abstract: [Objective] High hydrostatic pressure (HHP) could be synergized with Nisin in neutral food system to achieve commercial sterilization. The mechanism of synergistic inactivation by HHP treatment and Nisin was investigated at molecular level alongside ultramicroscopic structure. [Methods]Bacillus subtilis strain was subjected to HHP (100－500 MPa for 15 min) alone and in combination with Nisin (200 IU/mL) in PBS (pH 7.0). Membrane permeability was studied by fluorescent dye and ultraviolet absorption. Membrane lipid, protein and DNA changes were detected by Fourier Transform Infrared. Transmission Electron Microscope (TEM) was used to detect the morphological changes. [Results]In neutral food system, the combined effect of HHP and Nisin reduced the population of the bacteria by 2.5 log, while at the same time increasing the membrane permeability by 10%－60%. The physical state of membrane phospholipid molecules changed from liquid crystalline to gel state with a decrease in membrane fluidity. Treatments at elevated pressures were observed to cause protein denaturalization. Results from TEM demonstrate that cell content leaked owing to the synergistic effect. [Conclusion] This confirms that Nisin is an effective bactericide when combined with HHP in neutral food and that the two inactivation methods have a synergistic effect.

Abstract:Abstract: [Objective] The purpose of our study was to separate and identify a bacillus that could convert stevioside specifically. Then we identified the conversion product and studied the conversion capability of the bacillus. We also studied the enzyme with conversion capability and the conversion characteristic of the enzyme. [Methods] The bacillus was identified on the basis of morphology features and 16S rDNA sequence analysis. Phylogenetic tree was constructed to determine its taxonomic status. The product was detected and identified by high performance liquid chromatography and liquid chromatography-mass spectrometry methods. We use bacteria media directly to studied the conversion capability of the bacillus, and use resting cells, extracellular fluid and intracellular fluid to convert stevioside to determine the enzyme and made further study to learn its conversion characteristic. [Results] The 16S rDNA sequence of the strain had 99% similarity with Chryseobacterium sp., which was ultimately identified as Chryseobacterium sp.JH. The product of biotransformation was rubusoside and the enzyme that converts stevioside into rubusoside was intracellular enzyme. The conversion rate could reach 100%, obtained 5.7 g/L rubusodide solution after 48 h by bacteria media when the concentration of stevia glycosides was 10 g/L, including 7.2 g/L stevioside. When stevia glycosides was incubated with the enzyme for 12 h under the condition of pH 7.0, 45℃, initial intracellular enzyme liquid volume: transforming liquid volume 2: 5, stevia glycosides concentration 10 g/L, the conversion rate of stevioside reached 95.0%. It could reach 100% after 15 h. [Conclusion] The isolated strain JH was identified as Chryseobacterium sp.. It was a novel strain with high, specific ability to convert stevioside into rubusoside which had potential applications.

Abstract:Abstract: [Objective] The target of this study was to develop an engineered Sacchromyces cerevisiae strain to produce L-lactic acid efficiently using glucose as carbon source. [Methods] For construction of the strain CEN.PK2-1C[LDH], we integrated a LDH gene coding L-lactic acid dehydrogenase from Bovine into the genome of S. cerevisiae via homologous recombination and meanwhile knocked out a PDC1 gene coding pyruvate decarboxylase. As this, the carbon fluxes were led into L-Lactic acid. On the basis, we analyzed the Km value of these key enzymes to NADH and successfully over-expressed a NADH oxidase (nox) from Streptococcus pneumoniae into the cytoplasm for the construction of S. cerevisiae CEN.PK2-1C[LDH]-nox. [Results] Compared to initial strain, the yield of L-lactic acid in CEN.PK2-1C[LDH] fermentation liquor increased from 0 g/L to 15 g/L and the concentration of ethanol decreased from 27.3 g/L to 16.2 g/L. Furthermore, the concentration of L-lactic acid increased further to 20 g/L and the concentration of ethanol decreased to 8.2 g/L. [Conclusions] The results indicated that carbon metabolic flux was redistributed to efficient accumulation of L-lactic acid through two-sided control that heterologous expression of the gene LDH and decreasing the ratio of NADH/NAD+.

Abstract:Abstract：[Objective] To study the biological character and pathogenic effect of a lysozyme-like protein in Streptococcus pneumoniae. [Methods] The gene knockout mutant was constructed by the long flanking homology polymerase chain reaction. The mutant containing rescue plasmid to complement the lysozyme-like gene was also constructed. The differences in biology and pathogenicity in D39 wild strain, gene knockout mutant and gene knockout mutant containing rescue plasmid were observed to identify the functions of the lysozyme-like gene. [Results] Compared with the wild strain, the gene knockout mutant had the characteristics of slower growth, declining virulence, and obviously reduced capsule polysaccharide synthesis. Complement experiment showed when the rescue plasmid was transformed into the mutant strain, the mRNA level of hypothetical lysozyme-like gene in the gene knockout mutant containing rescue plasmid was higher than that of the wild strain .Although the levels of virulence and capsule polysaccharide synthesis could be partly complemented compared with those of the gene knockout mutant, but not reached the levels in the wild strain. [Conclusions] The lysozyme-like protein, a new Streptococcus pneumoniae virulence factor, may regulate the proliferation and the capsular polysaccharides synthesis of Streptococcus pneumoniae to affect expression of virulence.

Abstract:Abstract: [Objective] To identify the catalytic residues of mannanase AaManA from Alicyclobacillus acidocaldarius. [Methods] Based on the sequence alignment by ClustalX and ESPript and the structure information of GH-53 family, the possible catalytic residues were selected and mutated by overlap extension PCR. The protein of wild type and mutant were expressed in E. coli BL21(DE3) and ordinal purified by Ni-NTA affinity chromatography, gel-filtrate chromatography and ion-exchange chromatography. The purified protein was analyzed by thin layer chromatography (TLC) and the dinitrosalicylic acid (DNS) methods for enzyme assay. [Results] Seven mutants, E151A, E159A, E231A, C150A, E151Q, E231Q and double mutation E151Q&E231Q were successful constructed. Mutant E159A showed similar activities with wild type, and C150A mutation resulted in only a 3-fold reduction in the activities, but mutations E151A, E231A, E151Q, E231Q and E151Q&E231Q resulted in sharp decreases or loss in the activities, indicating that Glu151 and Glu231 play critical roles in AaManA activity. Furthermore, the presence of Glu151 at the C terminus of β4 and Glu231 at the C terminus of β7 was entirely consistent with the positions of the acid/base catalyst and the nucleophile catalyst of a GH-A enzyme, respectively. [Conclusion] By combining the results of TLC and enzyme assay of those mutants and the structural comparisons, it was confirmed that Glu151 and Glu231 fulfilled the roles of an acid/base catalyst and nucleophile catalyst in AaManA, respectively.

Abstract:Abstract：[Objective] In order to study the diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in shrimp farm sediment. [Methods] Total microbial DNA was directly extracted from the shrimp farm sediment. The clone library of amoA genes were constructed with β-Proteobacterial-AOB and AOA specific primers. The library was screened by PCR-restriction fragment length polymorphism (RFLP) analysis and clones with unique RFLP patterns were sequenced. [Results] Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from shrimp farm sediment were affiliated with Nitrosomonas (61.54%) or Nitrosomonas-like (38.46%) species and grouped into Nitrosomonas communis cluster、Nitrosomonas sp.Nm148 cluster、Nitrosomonas oligotropha cluster. All AOA sequences belonged to the kingdom Crenarchaeote except that one Operational Taxa Unit（OTU）sequence was Unclassified-Archaea and fell within cluster S (soil origin). AOB and AOA species composition included 13 OTUs and 9 OTUs. The clone coverage of bacterial and archaeal amoA genes was 73.47% and 90.43%. The Shannon-Wiener index, Evenness index，Simpson index and Richness index of AOB were higher than those of AOA. [Conclusion] These findings represent the first detailed examination of archaeal amoA diversity in shrimp farm sediment and demonstrate that diverse communities of Crenarchaeote capable of ammonia oxidation are present within shrimp farm sediment, where they may be actively involved in nitrification.

Abstract:Abstract: [Objective] Investigation of ammonia-oxidizing microorganisms (AOM) in natural environments is of great importance to understand global nitrogen cycling. However, little is known about the effects of dam constructions on the AOM community. We studied the diversity of the free-living and particle-attached AOM populations in the waters behind and in front of the Three Gorges Dam of the Yangtze River, and analyzed the possible correlation between the observed difference in the two fractions of AOM with the environmental parameters. [Methods] Two sampling locations near the Three Gorges Dam were selected: one behind and the other in front of the dam. Physicochemical profiles of waters at each location were measured, and the biomass in the waters was collected by filtration. The diversity of AOM in the collected samples was investigated by using an integrated approach including reverse transcription and clone library construction. [Results] The turbidity, dissolved oxygen, and redox potential of the water in front of the dam were higher than those behind the dam. The AOM population behind and in front of the dam was dominated by ammonia-oxidizing archaea, whereas the ammonia-oxidizing bacteria were not detected. The distribution of free-living and particle-attached AOA behind and in front of the dam was different: the particle-attached AOA behind the dam was more diverse than that in front of the dam, whereas the free-living AOA showed the opposite tendency; the difference between the fractions of AOA behind the dam was apparently higher than that in front of the dam. [Conclusion] The dominant AOA population did not show significant variation in the waters behind and in front of the dam, whereas the altered water dynamics resulted from the TGD construction may change the distribution of free-living and particle-attached AOA fractions in the waters behind and in front of the dam.

Abstract:Abstract: [Objective] To investigate the effect of Escherichia coli heat-labile enterotoxin (LT) B subunit (LTB) gene on canine parvovirus (CPV) VP2 gene vaccine. [Methods] The LTB gene was amplified by PCR from genomic DNA of E. coli 44815 strain. The VP2-70 fragment (210 bp) encoding major epitope of VP2 (70 amino acids) was amplified by PCR from a plasmid encoding VP2 gene. VP2-70 and LTB genes were inserted into the eukaryotic vector to construct VP2-70 gene, LTB gene and VP2-70-LTB fused gene vectors. The mice were immunized with VP2-70 vector, VP2-70-LTB fused vector, or VP2-70 vector plus LTB vector, respectively. The antibody titers at the different time were measured by using ELISA method. The spleen lymphocyte proliferation activity was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide（MTT）assay. [ Results ] The sequence of VP2-70 and LTB genes was identified. The recombinant VP2-70 and LTB proteins could be expressed in HEK293T cells in a secretory manner. The mice immunized with VP2-70 vector, VP2-70-LTB vector or VP2-70 vector plus LTB vector could generate the specific antibody against VP2 protein. The antibody titer immunized with VP2-70-LTB vector reached 1:5120 at 35 d post immunization, significantly higher than that of other two groups (P<0.01). For antibody isotype analysis, the IgG1 isotype antibody titers in all test groups were significantly higher than of IgG2a (P<0.01). The high-level spleen lymphocyte stimulation index was observed in the three test groups under the stimulation with Con A, higher than that in control groups (P<0.01). [ Conclusion] LTB gene could enhance the humoral immune response of CPV VP2 gene vaccine in mice.

Abstract:Abstract: [Objective] The purpose of this study is to produce new antibody molecules to neutralize the rabies virus specifically by the way of constructing and expressing the human anti-rabies virus scdsFv (disulfide stabilized single chain antibody) gene, and characterizing its bioactivity. [Methods] We obtained the sequence of variable region of heavy chain (VH) and variable region of light chain （VL) of RV monoclonal antibody SO57 from GenBank, and it respectively mutated into cysteine in the gene loci VH44 and VL100. The scdsFv gene was synthesized and inserted into a prokaryotic expression vector pET22b(+). Purified inclusion body scdsFv proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay. The binding activity of scdsFv was identified by ELISA and mouse brain tissues infected with rabies virus CVS strain. The relative affinity of scdsFv was measured by ELISA using thiocyanate elution. The capacity of the scdsFv to neutralize the rabies virus CVS strains was determined by fluorescent antibody virus neutralisation test (FAVN). The fusion proteins neutralized the CVS strain in a standard in vivo neutralized assay where the virus was incubated with the scdsFv molecules before intracranial inoculation in mice. [Results] The fragment genes of scdsFv to rabies virus were constructed successfully. ScdsFv protein was expressed in E.coli with approximate molecular weight of 30.0 kDa, which could be recognized by anti-His mAb. ELISA results demonstrated that scdsFv could bind antigen specificity. It was found that the strong reactivity of scdsFv to the smear of RV infected mouse-brain was demonstrated by IFA. As determined by FAVN with a reference serum, the titer of scdsFv was 41IU/mL. In addition, scdsFv could be 55.6% protection of mice against lethal challenge with rabies virus CVS. [Conclusion] The scdsFv can bind antigen specificity and has neutralization capacity to the virus in vitro and in vivo. The anti-rabies scdsFv is potential for application in rabies post-exposure prophylaxis.

Abstract:Abstract: [Objective] Our study is to analyse the coinfection effects of porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) in vivo on phagocytosis and interferon mRNA expression level of porcine alveolar macrophages (PAM). [Methods] Forty-eight 5-week-old healthy piglets were divided randomly into 4 groups of 12 (PCV2, PPV, PCV2/PPV and the control groups). The piglets in PCV2 group were inoculated oronasally with 3 mL of porcine circovirus type2 (PCV2, 105.61 TCID50/0.1 mL), PPV group with 3 mL of porcine parvovirus (PPV, 106.69TCID50/0.1 mL), PCV2/PPV group with 3 mL of PCV2 and 3 mL of PPV and the control group with 3 mL of cell culture medium, respectively. Three piglets from each group were sacrificed randomly on 3, 7, 14 and 35 day post infection (dpi) and porcine alveolar macrophages (PAM) were collected to detect viability, phagocytotic capabilities and α1- and γ-interferon (IFN-α1 and IFN-γ) mRNA levels of PAM. [Results] The viabilities of PAM from PCV2 group and PCV2/PPV group became weaker than that of control group during the period of 3–14 dpi but they were similar to that of control group on 35 dpi; there was no significant difference between the viability of PCV2/PPV group and that of PCV2 group（P＞0.05）. The phagocytotic capabilities of PAM from three virus infection groups were lower than that of control group (P<0.01), among which that of PCV2/PPV group descended more drastically. IFN-α1 and IFN-γ mRNA levels in PAM from PCV2/PPV group were significantly lower than those of PCV2, PPV and control groups (P<0.01). [Conclusion] PCV2/PPV co-infection did not cause further decline of PAM viability but strongly weakened phagocytosis and constantly lowered IFN (IFN-α1 and IFN-γ) mRNA expression levels of PAM.

Abstract:Abstract: [Objective]n order to select Aspergillus japonicus mutant strains that express high-yield of glycerol oxidase, we constructed a mutant library of Aspergillus japonicus that mediated by Agrobacterium tumefaciens. [Methods]Tri-relative hybridization experiments were performed to transfer binary vector pBI-hphII into Agrobacterium tumefaciens EHA105, which was used as the infective strain and Aspergillus japonicus was used as the host strain. We established the transformation system of Aspergillus japonicus mediated by Agrobacterium tumefaciens and constructed a mutant library. At the same time, we analyzed the factors affecting the transformation efficiency such as the concentration of Agrobacterium tumefaciens, adding and not adding Acetosyringone, co-culture time, co-culture temperature and so on. [Results]The results of PCR detection and Southern analysis showed that the T-DNA was integrated into genome of the Aspergillus japonicus. All of the nine randomly selected transformants were quite stable after ten rounds of successive cultures. [Conclusion]The transformation system is a good basis for selection of Aspergillus japonicus mutant strains which give a high-yield of glycerol oxidase.

Abstract:Abstract: [Objective] The aim of study was to investigate bacterial diversity of Populus Euphratica forest in the hinterland of Taklimakan desert. All the isolateds were be used as inoculants for silage and biofertilizer. [Methods] Strains were isolated by culture-dependent method. Gram staining, NaCl tolerance, enzyme activity (including amylase, esterase, cellulase) were determined by strand methods. Phylogenetic trees based on 16S rRNA gene sequences were constructed by using the neighbour-joining. [Results] A total of 27 strains were obtained. Phylogenetic analysis of the bacterial 16S rRNA gene showed that all isolates fell into one of the following four bacterial lineages: Actinobacteria(16 strains), Proteobacteria(4 strains), Firmicutes(6 strains) and Bacteroidetes(1 strains). Gram staining indicated that 5 strains were gram-negative and the others were gram-positive. Among these, 15 strains showed amylase activity, 9 strains showed esterase activity and 9 strains showed cellulase activity. All strains growth occurred at in presence of 2% NaCl, 22 strains growth occurred at in presence of 5% NaCl and only 1 strain tolerated up to 15% NaCl. [Conclusion] The bacterial population diversity is abundant in soil of Populus Euphratica Forest in the hinterland of Taklimakan Desert, which is worthy of futher investigation.

Abstract:Abstract: [Objective] To find specific polypeptides that bind porcine reproductive and respiratory syndrome virus (PRRSV) with high affinity and inhibit replication of PRRSV, we screened ligands on intact PRRSV virion by phage display library. [Methods] The purified PRRSV was coated and then reacted with random peptide library displaying 12 amino acids fused on protein Ⅲ of M13 phage. The selected peptides for target binding were assayed by ELISA after 3 rounds of biopanning and measured by 50% tissue culture infection dose. The positive clones with high affinity were used for automated sequencing, and the amino acid sequence of polypeptide displayed on phage was deduced. Synthesis of fluorescein isothiocyanate labelled polypeptide and establish a method for detection of PRRSV. [Results] The enrichment was shown by ELISA after 3 rounds of biopanning and 17 positive clones bound to PRRSV with high affinity. Sequencing of the genes encoding these peptides in positive clones show some of conserved motifs. Two positive clones inhibited the replication of PRRSV in Marc-145 cells in vitro and decreased PRRSV TCID50 from 10–7.3/0.1 mL to 10–3.2, 10–3.6/0.1 mL respectively. The fluorescein isothiocyanate labelled peptide was able to detect PRRSV at the concentration of 5 mg/L. [Conclusion] Positive clones against PRRSV can be selected from phage display peptide library and so provide a potential tool for highly sensitive diagnostic kits and novel antiviral agents.

Abstract:Abstract: 【Objective】 In order to get a rapid specific diagnostic reagent for subgroup A Avian Leukosis Virus detection.【Methods】The Avian Leukosis Virus Subgroup A(ALV-A) SDAU09E1 strain was inoculated into DF1 cells, an ALV-A-gp85 DNA fragment of 1023bp was amplified from infected cells and inserted into PET-32a(+) plasmid at the location between restriction endonucleases BamHⅠand NotⅠsites. The recombinant plasmid PET-SDAU09E1-gp85 was transformed into E coli.BL21 (Rosetta) for gp85 gene expression. Then we used the purified recombinant fusion protein to immunize 6 weeks old Kunming white mice, and the antiserum were prepared. 【Results】The recombinant ALV-A gp85 fusion protein with a molecular weight of 52.8kDa demonstrated a good antigenecity. Mon-specific serum produced by vaccinated mice came out reactive with subgroups A and B ALV(ALV-A and ALV-B but not subgroup J ALV) by the indirect immunofluorescence (IFA) method. 【Conclusion】This was the first time to demonstrate a mono-specific antiserum specific to ALV-A and ALV-B,it could be used for differential diagnosis of exogenous ALV infections in CEF cultures when in complement with ALV-J specific monoclonal antibodies. Chickens in our country are now distressed by both classic ALV-A/B and emerging ALV-J, making differential diagnosis necessary, so studying this reagent has high practical value.