Abstract:A Streptomyces rameus strain with high yeild in xylanase activity was identified. Its capacity to produce xylanase and the characteristics of its hydrolysates were also investigated. A new strain of Streptomyces sp. L2001 was isolated from soil samples and was identified using morphological, cultural, physiological and biochemical characterisctics. The total length of 16S rDNA fragment was 1429 bp through PCR amplification. Our results showed that 16S rRNA sequence of Streptomyces sp. L2001 homologied up to 99.16% with that of Streptomyces rameus NBRC3782. Combining the traditional characteristics from physiological and biochemical tests, the strain L2001 was identified as Streptomyces rameus. As the firstly-reported strain to be capable of producing xylanase, its maximal yield could reach to 842.0 U/mL within 6 days’ cultivation under submerged fermentation condition. Using HPLC analysis, the total contents of xylobiose, xylotriose and xylotetraose constituted 93.5% of the hydrolysates of the xylanase from Streptomyces sp. L2001. Our results suggested that the xylanase is applicable to industrial production of functional xylo-oligosaccharides.

Abstract:A strain producing cold-active raw starch-digesting glucoamylase was isolated from Bohai Bay’ samples. The strain RS01 was identified as Aeromonas sp. based on the morphological, physiological characteristics and 16S rRNA sequence. The foundation on the enzymatic properties of partially purified Cold-active RSGA explained that the maximum activity of the enzyme was exhibited at 30°C and pH 5.4. The enzyme thermal stability was poor. The glucose was identified by the TLC, which proved that the strain RS01 has the ability to produce cold-active raw starch-digesting glucoamylase.

Abstract:The single factor experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium and conditions of the biocontrol agent of Bacillus cereus AR156. The results indicated that the constituents of medium, the filling volume of shaking flask, the rotation speed and the temperature were the major factors that affect the yield of AR156. The composition of maltose 0.25%, maizena 0.5%, soybean flour 0.5%, tryptone 0.5%, CaCl2·2H2O 0.05%, MnSO4·H2O 0.05% and K2HPO4 0.1% were selected to be the medium formula, and the fermentation conditions were the filling volume 200 mL in 1L shaking flask, inoculum 5%, 28°C, shaking at 200 r/min for 48 h, and initial pH at 7.0. Under the optimized fermentation conditions the production of spores was up to 1.03 × 109 CFU/mL, and the rate of spores was more than 97%.

Abstract:The effects of different CHCl3 concentrations treatment on fermentative H2 production and microbial diversity of anaerobic sludge were investigated. At CHCl3 concentration of 0.050%, the cumulative H2, maximum H2 yield, VFA concentration and total sugar degradability all reached the maximum and were 639 mL, 1.71 mol H2/mol consumed glucose, 2880 mg/L and 85%, respectively. The microbial diversity and community structure of sludge samples with different CHCl3 concentrations treatment were analyzed by applying PCR-DGGE. The results showed that 4 bacteria clones belonged to Clostridia, 2 bacteria clones belonged to Acidobacteria and δ-proteobacteria, respectively, and other 4 bacteria clones were uncultured bacterium. All 4 microbes that belonged to Clostridia were H2-producing bacteria (HPB) and bacteria of band 7 might belong to HPB. The optimum microbial community structure for H2 production was observed in sample C3, which mainly contained Megasphaera sueciensis, Megasphaera paucivorans, Clostridium cellulosi, Clostridium sp. and uncultured bacterium.

Abstract:Some numerically dominant colonies, with creamy and nondiffusionable morphology, were isolated from lead-zinc tailings and their phylogenetic and physiological characteristics were determined. This type of colony represented approximate 50% of cultivable bacteria of tailings. Phylogenetic tree constructed from about 500 bp of 16S rRNA gene of 60 isolates and their relatives indicated that, these strains belonged to the member of Arthrobacter genus and were divided into several phylogenetically distinct groups. Three representatives from each group, BS11, BS20 and AS19, respectively, were sequenced approximate 1440 bp of 16S rRNA gene and their phylogeneic positions were further determined. Strain BS11 was phylogenetically close to A. histidinolovorans and A. nicotinovorans, BS20 close to A. chlorophenolicus, and AS19, to A. aurescens and A. ilicis. Among 39 carbon sources tested, three strains could strongly utilize 15 and weakly utilize 12 carbon sources. All of three strains showed high resistance to Pb(NO3)2, ZnSO4, CdCl2, CuSO4 and CoCl2, but relatively sensitive to ampicilin, streptomycin, kanamycin and rifimpicin. Our data suggested these strains may be potential novel species.

Abstract:Fourty one plant tissue samples, including root, stem, leaf and hypocotyl were collected from 8 kinds of eumangrove and 5 kinds of semi-mangrove at Shankou Mangrove Nature Reserve in Guangxi Province. These samples were grinded into little pieces after surface sterilization, and then seeded into the isolation media containing chitin, humic acid or xylan as the main nutrition and energy source. As a result, 118 actinobacterial strains were purified from these plant tissue samples. These isolates were screened using 6 models for screening new antibiotics, and the fermentation broths from 77 strains exhibited activity on at least one screening model, the positive rate among the isolates is 65.3%. Based on the morphological characteristics, chemotaxonomic and 16S rRNA gene analysis, 77 isolates were identified as Streptomyces spp. (44), Micromonospora spp. (25), Saccharothrix spp. (3), Nocardia spp. (3), Nocardiopsis sp. (1) and Lentzea sp. (1). Strain I07A-01824, which showed strong antibacterial activity against the test strain Enterococcus faecalis HH22 (a multi-resistant clinical isolate) and positive activity on the screening model of statins-like antihyperlipidemics, was identified as a member of a known species Streptomyces albidoflavus. This study demonstrates that an unexpected diversity of actinobacteria colonizes inside the mangrove tissues, and these endophytic actinobacteria are promising sources of new natural secondary metabolites with physiological activities.

Abstract:The technique of using the pupae of Chinese tussah, Antheraea pernyi, as a surrogate host to artificially rear Chouioia cunea has been playing an important role in the biological control of the fall webworm, Hyphantria cunea, in Liaoning, Beijing, Tianjin, Shanghai, Hebei, Shandong Provinces or Municipalities and other places of China. Bacterial diseases of Chinese tussah pupae are the main obstacle to mass rearing C. cunea. Based on the fact that the tissue in some pupae turned pink after liquefying during rearing C. cunea, which is a typical symptom of an unknown bacterial disease, a bacterium was isolated and purified, and C3 was obtained. C3 was identified as a strain of Serratia marcescens by conventional techniques, Biolog system and 16S rRNA analysis, and was determined as the pathogenic bacterium causing the disease to Chinese tussah pupae by a test based on Koch’s postulates. The identification characteristics were described of the disease in its incidence in the process of rearing C. cunea.

Abstract:To analyze the dynamic changes of LAB (Lactic acid bacteria) during a 15-day ensilage of corn and the effect that came from inoculants. Plate isolation and 16S rRNA gene homology methods were used to take count of LAB numbers and analyze the population changes during ensilage of corn. One hundred and fifty two LAB were identified as species of Lactobacillus and Pediococcus which were isolated from fermented corn, whereas 86% colonies were identified as Lactobacillus by 16S rRNA gene sequence analysis. The maximum numbers of control group in fermented corn achieved 2.1 × 106 CFU/g and the second experimental group in fermented corn achieved 5.5 × 107 CFU/g after a week. It accelerated growth of Lactobacillus when microbial additive was added. The initial bacteria during ensilage of corn were Pediococcus and Lactobacillus, the dominant bacterium was Lactobacillus in the later phase.

Abstract:To establish a detection method of food borne Proteeae pathogens, we compared different enrichment broths and different selective agars on Proteeae pathogen growth, correlated with the biochemical characteristics of the pathogens. The results showed that EE enrichment broth was more efficient than GN broth, and the selective agars were EMB agar plus SS agar (or MacConkey agar). The preliminary screen methods were Gram-stain reaction and phenylalanine deaminase reaction. The Proteeae pathogens were finally confirmed by other biochemical tests. This method was sensitive and had a broad scope of detection, could provide experimental basis for new standard.

Abstract:Strain G-41 was isolated from the soil. The strain was identified as Bacillus by 16S rRNA method. Growing in fermentative medium, the strain can produce high-yield alkaline protease (1.7×104 U/mL). We delt with the original strain (G-41) by the heavy-ion irradiation and obtained the mutant G-41-68. The mutant G-41-68 was again treated by the heavy-ion irradiation. We screened a high-yield alkaline protease producing strain 15Gy-54 from many of mutants and its enzyme activity reached to 6.22 × 104 U/mL. Compared with the original strain, the enzyme activity of mutant strains G-41-68 and 15Gy-54 increased by 1.58 and 2.65 times, respectivity. The fermentation conditions of the mutant strain 15Gy-54 were optimized and its enzyme activity further increased, reaching to 7.18 × 104 U/mL. The mutant’s optimum conditions for enzyme production consisted of 1% tryptone, 0.5% yeast extract, 5% lactose, 0.4% Na2HPO4·12H2O, 0.03% KH2PO4, 0.1% Na2CO3, 4 × 10-3 mol/L MgSO4, the initial pH 8.0, shaking culture for 42-48 h, at 41°C. These results showed that the heavy-ion irradiation is an effective method for microbe mutagenesis.

Abstract:Variation in gene expression of Escherichia coli (E. coli), treated to micro-gravity, is analyzed to discover the change of microbial metabolism and the adaptation mechanism in microgravity using Microarray Gene Expression Profiles and Mass Spectrometry Analysis. In this article the strain was cultured in a rotator for several days and the mutant with advanced logarithmic phase in growth curve was screened and selected for Microarray Gene Expression Profiles and Mass Spectrometry Analysis. One hundred and fourteen differentially expressed genes are shown in Microarray Gene Expression Profiles of the mutant, and among them 99 were up-regulated expressions. The up-regulated expressions mainly appeared in ABC transporter, sugar metabolism, 3-carboxylic acid metabolism, phosphotransferase system, nucleic acid metabolism, lipid metabolism, etc. The result was verified through protein analysis with mass spectrometry. Suggesting that microgravity processing may accelerate microbial growth, and the advanced growth of the strain resulted from the up-regulated gene expression. The strain can adapt to spatial environments by affecting genes related to microbial metabolism.

Abstract:Eupenicillum sp. E-0509-2 was used as an original strain to carry out mutagenetic breeding adopting UV, NTG and UV + NTG, respectively. The spore suspension of E-0509-2 was treated by UV irradiation for 60 seconds, followed with 3.0 g/L NTG for 20 minuites under magnetic stirring. After further screening experiments on fermentation, a mutant Eupenicillum sp. E-UN41 which was able to produce mizoribine with high yield and could be stably passed on genetics was eventually found. The Mizoribine yield of Eupenicillum sp. E-UN41 was enhanced more than 13 times higher than that of the original strain.

Abstract:An open reading fragment, SSU2099, encoding pilus gene from srtBCD pilus island was identified based on bioinformatics analysis of the genome sequence of 05ZYH33 and alignment with relative protein family. The target DNA fragment was amplified using a pair of primers specific to SSU2099 gene. SSU2099 gene was cloned into prokaryotic expression plasmid pET32a subsequently, and the recombinant protein was expressed and purified. The mice anti-SSU2099 serum was obtained by immunizing mice with recombinant SSU2099 protein, and the titer of the serum was analyzed by ELISA. Animal test showed that vaccinating mice with recombinant SSU2099 confer a significant protection, which indicated that SSU2099 can serve as a novel vaccine candidate. Further studying of the localization and function of SSU2099 may adds novel insights into the role that srtBCD pilus island plays in pathogenicity of Streptococcus suis 2.

Abstract:Borrelia Burgdorferi is the causative pathogens for Lyme disease. A loop-mediated isothermal amplification (LAMP) rapid assay was developed for genotyping Borrelia borgdorferi based on the outer surface protein A (OspA) gene. All three different genotypic isolates including B. burgdorferi sensu stricto, B. afzelii and B. garinii, distributed mainly in China were detected and genotyped successfully using the LAMP method. The genotyping of Borrelia burgdorferi would provide evidence for treatment of patients with different clinical symptoms and for control of Lyme disease.

Abstract:A preliminary study on the antagonistic protein produced by Burkholderia pyrrocinia JK-SH007 was carried out. The results showed it was stable to heat, not sensitived to proteinase K and partially sensitive to alkali. Cell culture was obtained after centrifuging and filtering (0.22 μm) of ferment liquid of B. pyrrocinia JK-SH007 by shake-flask fermentation. The crude proteins were obtained by fractionation with (NH4)2SO4 (20%-60%). Active proteins(A-Ⅱ-2) were obtained after purification of the crude proteins by 3.5 kD dialysis bag dialysis, cold acetone precipitation (-20°C), Sephadex G-50, DEAE-Sephadex A-25 aion-exchange column chromatography. A-Ⅱ-2 were shown three bands by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins showed good inhibition activity to three pathogens Cytospora chrysosperma, Phomopsis macrospore and Fusicoccum aesculi which caused poplar canker.

Abstract:To clone, express and characterize the HA and NA Protein of avian influenza A virus H9N2. On the basis of successful clone the full length HA and NA gene and sequence analysis of avian influenza A virus H9N2, we were ligated part of the gene into pET32a (+) and full of the gene into pGEX4T-1. An expression vector pET32a (+)/HA (cut), pET32a (+)/NA (cut), pGEX4T-1/HA, pGEX4T-1/NA were constructed and expressed in E. coli BL21/rosetta induced by IPTG. Recombinant protein was purified through affinity chromatography column. Western Blotting and ELISA were used to determine the antigenic of the recombinant protein. The recombinant capsid gene can be overexpressed in E. coli. SDS-PAGE result showed that the gene could express product as same as we expect. ELISA and Western Blotting result showed that the recombinant protein has good antigenic. The HA and NA protein of avian influenza virus H9N2 has been successful cloned and expressed, which could be useful for developing diagnose reagents or vaccine of H9N2.

Abstract:As an opportunistic pathogen, Pseudomonas aeruginosa can produce biofilm and pyocyanin, which play a critical role in its pathogenesis. This study aims to elucidate the function of the Arr gene in formation of biofilm and production of pyocyanin in Pseudomonas aeruginosa. Using the chromosome DNA of Pseudomonas aeruginosa as template, we first cloned the Arr gene by PCR. With the insertion of gentamycin resistance cassette (aacC1), the mutant PA-AG has then been constructed by homologous recombination. The formation of biofilm is determined by staining with crystal violet, and production of pyocyanin is detected with spectrophotometric method. In KMB or LB medium, biofilm formation of the parental strain PAO1 is about 2 folds higher than that of the Arr mutant PA-AG. The biosynthesis of pyocyanin in the Arr mutant PA-AG is about 2.5 folds higher than that in the parental strain PAO1. These results indicate that the Arr gene negatively controls the pyocyanin biosynthesis, but Arr could exert some positive effect on the biofilm formation. It is suggested that regulation mediated by the Arr gene on biofilm and biosynthesis of pyocyanin in Pseudomonas aeruginosa is specific and different.

Abstract:The optimization of fermentation medium is important for improving microbial production or quality in fermentation industry. Experimental design and optimization techniques play an important role for optimizing medium. In this paper, we reviewed the methodologies and techniques used in optimization of microbial medium, including one-factor-at-a-time design, factorial design, uniform design, response surface optimization (RSM), artificial neural network (ANN) and genetic algorithms (GA). Meanwhile, the methodologies and techniques were analyzed and evaluated comprehensively.

Abstract:A vast number of secondary metabolites are produced in Streptomycetes under the tight control by many regulators, including two-component regulatory systems. Two-component regulatory systems with high diversity are widely distributed in Streptomycetes and perform positive or negative regulation by different and complex modes during secondary metabolites biosynthesis. Here, we reviewed some research advances on these systems involved in secondary metabolites accumulation in streptomycetes, mainly focusing on the clarification of their action modes. We also prospected the research trends in this field and application of two-component regulatory systems in the metabolic engineering of natural products.

Abstract:In this paper, the character of the developing virology and virology course is discussed. Based on the traditional teaching experience and current developing trend, the authors give suggestion in teaching material, chapter arrangement and experiment course teaching. The practice of virology in Wuhan University is also introduced.

Abstract:Lipopolysaccharide (LPS) is the major component of the cell wall of Gram-negative bacteria, and is also associated with the pathogenicity of bacteria closely. But it has been a major mistaken ideas for a long time to define LPS as endotoxin in the teaching of Medical Bacteriology. And which might prevent students to study and understand the pathogenicity of bacteria correctly also. So here we offer some initial ideas for discussion.

Abstract:Some reforms in teaching content, teaching modes and examination of the environmental microbiology course for students (including senior undergraduate and graduate students majored in biotechnology) were presented. The reforms include how to renew teaching outline, chose new textbooks in English and Chinese, renew teaching contents, and the means to encourage active learning and examine the capacities of students.

Abstract:Rapid and efficient inactivation of the target gene on Escherichia coli chromosome is the precondition and groundwork of researches on metabolic engineering. In the present study, we demonstrated a multiple gene inactivation approach in E. coli mediated by Red recombination and Xer recombination. The chromosomal genes, ackA-pta and pps, in a wild type strain E. coli CICIM B0013 were inactivated by this method indicating that dif sites can be reused to inactivate multiple chromosomal genes with no antibiotic resistance selectable marker remain. Furthermore, this method has high recombination efficiency and simplified steps.

Abstract:We purified the McAbs and then determined the subclasses, activity and specificity of the Clavibacter michiganensis subsp. nebraskense McAbs (4H4 and 4G12). We compared the detection sensitivity between indirect ELISA and double antibody sandwich ELISA (DAS-ELISA) and utilized both of them on the detection of the corn bacteria. Titers of both McAbs in 0.4 g/L were 1 : 256000, subclasses of 4H4 belongs to IgG2a while 4G12 belongs to IgG2b. No cross reactivity was found within 16 bacterial strains tested from corn and other plants by ELISA. The minimun detection limit of the bacteria in the seed suspension with DAS-ELISA was 1.0 × 109 CFU/L. A specific and sensitive DAS-ELISA for detection of the C. michiganensis subsp. nebraskense was developed.

Abstract:

Abstract:C57BL/6J WT and mice deficient in MyD88 (MyD88 KO) were inoculated intravaginally with 1 × 104 IFUs of live C. muridarum organisms. Half mice of each group were reinfected on day 54 after primary infection. Vaginal swabs were taken every 3 or 4 days to monitor live organism shedding. On day 80 after the primary infection, mice were sacrificed, and the vaginal tract was isolated for pathology. The spleen cells were collected and IL-4, IL-5, IL-17 and IFN-γ were detected by ELISA in the spleen cells culture supernatant after restimulated by MoPn EB. The titers of different Ab isotypes were measured in mice serum by Indirect Immunofluorescence Assay. The Chlamydia shedding time of MyD88 KO mice was similar to WT. Not only the gross appearance of the isolated genital tracts, but also the dilation and inflammation scores under microscope showed that the genital tract pathology from the MyD88-deficient mice was much more severe than WT after primary infection. The results of Th2 (IL-4 & IL-5), Th1 (IFN-γ) and Th17 (IL-17) cytokines from the in vitro restimulated splenocyte culture supernatant showed that MyD88 deficient mice produced significantly lower levels of IFN-γ and IL-17 but much higher levels of IL-4 and IL-5 than WT mice either after the primary infection or reinfection with Chlamydia. There were no significant differences in Ab isotype levels between the two tested groups. However, the ratio of MoPn specific serum IgG2a/IgG1 in MyD88-deficient mice was less than 1 and significantly lower than that in WT mice. MyD88 is dispensable for protective immunity but required for inflammatory pathologies.

Abstract:

Abstract: