Abstract:Abstract: Enterobacter sakazakii is an important food-borne pathogen that causes life-threatening meningitis and necrotizing colitis in neonates and children. In 2008, Enterobacter sakazakii has been reclassified as five species in a new genus, Cronobacter gen. nov. within the Enterobacteriaceae. There is the variation in virulence between species in the new genus. OmpA of Cronobacter gen. nov. plays a critical role in attachment to their host cell and persistence within macrophages . Disruption of the tight junction significantly enhances the efficiency of invasion. Specific probiotic strains and their combinations counteract adhesion of Cronobacter gen. nov to intestinal mucus. At present, very limited information is available regarding the pathogenesis of Cronobacter gen. nov. Further detailed mechanism studies on the pathogenicity are warranted.

Abstract:Abstract: Microbial fuel cells (MFCs) are bio-electrochemical reactors that have the capacity to convert chemical energy of biodegradable organic chemicals to electrical energy, and developed rapidly in the past few years. With an increasing concern for energy crisis and environment pollution, MFCs has became a promising technology in the researches of environment pollution treatments and biology electricity. In this paper, we offered a comprehensive review of the recent research progress of MFCs in environment pollution treatment, includes denitrification, desufurization, organic pollutants degradation, heavy metal reduction and landfill leachate treatment. Also, we pointed out the challenges and problems which were bottle necks for a wide application of MFCs and the potential future development.

Abstract:Abstract: [Objective] The interaction between plant growth promoting rhizobacteria (PGPR) and plants can be unstable, PGPR with PGP activities may be well adapted to particular soil environment. Based on this, we isolated and identified PGPRs from different rhizosphere soils according to their multiple mechanisms. [Methods] Preliminary screening of PGPRs under the premises of PGPR may having the abilities of N2-fixing, phosphate and potassium solubilization, and resistance against six common pathogenic fungi as well as rhizosphere colonization. After that, multiple PGP activities were detected in vitro. Finally, PGPRs were classified and identified by combining physiological and biochemical tests and 16S rRNA gene sequence analysis. [Results] Fourteen strains having various mechanisms of PGP activities such as NH3, IAA, HCN, siderophore, antibiotics production, phosphate and potassium solubilization, and N2-fixing were isolated from different rhizosphere soils in Yangzhou and Yancheng, Jiangsu province. These 14 isolates could be identified as Pseudomonas (7 isolates), Paenibacillus (3 isolates), Bacillus (2 isolates), Burkholderia (1 isolate) and Erwinia (1 isolate). [Conclusion] Isolates with multiple PGP activities can also be rhizospheric competent, able to survive and colonize in the rhizosphere, providing promising isolates for PGPRs combination to resolve the challenges in field application of PGPR.

Abstract:Abstract: [Objective] The aim of this study was to investigate actinobacterial diversity in Chilean marine sediments. [Methods] Actinobacterial diversity in these sediments was investigated by selective isolation method, culture-independent method and phylogenetic analysis based on 16S rRNA gene sequences. Six selective media were used to isolate actinomycetes from sediment samples. The primers for the class Actinobacteria were used for Actinobacterial 16S rRNA gene amplification and then a clone library was constructed for the sediment sample btt. Twenty-two strains with different culture characteristics and 59 clones from sample btt were selected for 16S rRNA gene sequences analysis. To determine requirement for seawater each strain was grown on oatmeal agar prepared with deionized water and with seawater, respectively. Strains were screened for antibiotic activity against bacteria and fungi. [Results] In total 328 actinomycetes were obtained. Twenty-two strains which were selected belonged to Streptomyces, Micromonospora, Polymorphospora, Aeromicrobium and Brachybacterium. Fifty-nine clones (40 OTUs) were sequenced, and 60% OTUs belonged to Actinobacteridae, Acidimicrobidae and Rubrobacteridae. The other 40% OTUs, which formed several distinct clades in phylogenetic tree among phylum Actinobacteria may represent new taxonomical groups. 50% of the 47 sea water dependant strains and nineteen strains out of the above 22 strains exhibited antimicrobial activity. [Conclusion] There was abundant actinobacterial diversity in the marine sediments of Chile, and the result implied that there were large numbers of unknown actinobacterial groups in the sediments. Actinomycetes from Chilean marine sediments had the potential of producing bioactive secondary metabolites.

Abstract:Abstract: [Objective] The aim of this study was to screen straw-cellulose degrading microorganisms and to investigate their degradation ability of straw-cellulose. [Methods] The methods used to screen the high effect straw-cellulose degrading microorganism included the traditional isolation methods of straw-cellulose degrading microorganism such as holes observation method on filter paper sheet, disintegration test of filter paper scrip, hydrolysis spot diameter measurement method of CMC-Na, weight lose assay method of straw, measurement method of cellulose decomposition rate, measurement method of extracellular enzyme activity. [Results] We isolated 3 fungi with cellulose degrading ability, of which 98MJ was identified as Penicillium oxalicum, W3 as Trichoderma sp., and W4 as Penicillium expansum. Strain W4 possessed high straw-cellulose degrading ability with straw-cellulose degrading rate of 56.3%, cellulose 59.06%,, hemicellulose 78.75% and lignin 33.79% in 10 days. [Conclusion] Strain W4 was a cellulase-producing strain with broad development potential.

Abstract:Abstract: [Objective] Ultrastructural alteration and hydrogen peroxide (H2O2) localization were examined in Xanthomonas under cellular injury using transmission electron microscopy. [Methods] Histochemical methods were used in the present study. [Results] Intriguingly, the injury led to presence of an additional location of H2O2 accumulation within the cells. There was an association between the frequency and size of the additional location of H2O2 accumulation and the degrees of injury. Furthermore, an additional ultrastructure, mesosomes, was also present in injured cells. The frequency and size of mesosomes also increased with the increasing degrees of injury. [Conclusion] Result of multiple linear regression showed that the size of mesosome plays as a key factor in the quantity of excess H2O2 accumulation in bacteria under cellular injury. Linear correlation was confirmed between quantity of excess H2O2 accumulation and the size of mesosome in injured cells. This finding intensely indicated that mesosomes are just the additional location of H2O2 accumulation in cells under cellular injury. The excess H2O2 accumulation in mesosomes should be a positive regulatory mechanism in bacteria under cellular injury.

Abstract:Abstract: [Objective] Our study aimed at screening and identifying a specific bacterium capable of degrading stevioside. We also studied the conditions of enzyme production and stevioside conversion. [Methods] Taxonomic group of the strain was confirmed by physical characterization and phylogenetic analysis by 16S rRNA gene sequence analysis and phylogenetic tree construction of the strain. The optimum conditions of enzyme producing and stevioside degrading were studied by single factor and multi-factor statistical analysis. Degradation product was detected and identified via liquid chromatography-mass spectrometry. [Results] Based on the result of 16S rRNA gene sequence analysis, the strain named J2 shares 100% sequence identity with the sequence of the Bacillus megaterium. The activity of beta-Glucosidase produced by this Bacillus megaterium strain was up to 779.68 U/ml with 4% maize starch, 1% defatted soybean, 0.04% MgSO4 and 0.2% stevioside as culture medium when fermented under the condition of pH 7.0, 37℃, 220 r/min and 10% inoculum for 36 h. The results of conversion showed that 10 mg/ml stevioside can be converted to steviolbioside by 74% after 3 days which has been identified by LC-MS. The ratio of rebaudioside A and stevioside was increased to 0.99 compared to original solution 0.38, which lead to 160.5% increasement of rebaudioside A in the relative amount. Stevioside can be converted completely after 5 days. [Conclusion] The isolated strain J2 was identified as Bacillus megaterium. It was a novel and safe strain with high, specific conversion stevioside to steviolbioside ability.

Abstract:Abstract: [Objective] To clone, express and characterize a novel esterase from marine sediment microbial metagenomic library. [Methods] Using esterase segregation agar containing tributyrin, we obtained esterase positive fosmid clone FL10 from marine sediment microbial metagenomic library. This fosmid was partially digested with Sau3A I to construct the sublibrary, from which the esterase positive subclone pFLS10 was obtained. The full length of the esterase gene was amplified and cloned into the expressing vector pET28a, and the recombinant plasmid was transformed into E. coli BL21 cells. We analyse the enzyme activity and study the characterization of the esterase after its expression and purification. [Results] An ORF (Open Reading Frame) of 924 bp was identified from the subclone pFLS10. Sequence analysis indicated that it showed 71% amino acid identity to esterase (ADA70030) from a marine sediment metagenomic library. The esterase is a novel low-temperature-active esterase and had highest lipolytic activity to the substrate of 4-nitrophenyl butyrate (C4).The optimum temperature of the esterase was 20 ℃, the optimum pH was 7.5. The esterase in this study had good thermostability at 20 ℃ and good pH stability at pH8－10. Significant increase in lipolytic activity was observed with addition of K+ and Mg2+, while decrease with Mn2+ etc. [Conclusion] We obtained the novel esterase gene fls10 from the marine sediment microbial metagenomic library. The esterase had good thermostability and high lipolytic activity at low temperature and under basic conditions, which laid a basis for industrial application.

Abstract:Abstract: [Objective ] To clone and identify prenyl transferase gene from Metarhizium anisopliae and to understand the gene structure and expression. [Methods ] Using Switching Mechanism At 5' end of RNA Transcript (SMART) method, we isolated the full length cDNA sequence and DNA sequence. ?Then we used quantitative RT-PCR analysis of the gene expression levels at different stages of colonization of host hemolymph by M. anisopliae. [ Results] The Mpt gene had two exons and one intron and the CDS was 1026 bp which encoded a protein with 341 amino acid residues; qRT-PCR analysis showed that the gene expression levels were significantly different, especially highly up-regulated at the late stages. [ Conclusion] The Mpt gene was successfully cloned from M. anisopliae for the first time and the gene had the characteristic of high expression levels at the late stages.

Abstract:Abstract: [Objective] To study the interaction of streptococcal collagen-like protein 1 (Scl1) of M41-type group A streptococcus (GAS) ATCC12373 with low-density lipoprotein (LDL). [Methods] We cloned, expressed and purified the recombinant proteins rScl1 and its V region rScl1-V, designated as C176 and C176V, derived from Scl1.41 of M41-type GAS. The binding of rScl1 to LDL was detected with affinity chromatography-binding assay, Western blot analysis and enzyme-linked immunosorbent assay (ELISA), and whole cell binding assays were used to detect the interactions of whole GAS cells with LDL. [Results] The results demonstrated that C176 and C176V could specifically bind purified LDL, and M41-type GAS cells expressing native Scl1 could bind LDL whereas M6-type GAS could not. [Conclusion] The Scl1 of M41-type GAS specifically binds LDL.

Abstract:Abstract: [Objective] The pine wood nematode（PWN）, Bursaphlenchus xylophilus, which collaborates with its associated bacteria to form ecosystem and has interaction among them, is the pathogen of pine wilt disease. This study focused on revealing the bacterial diversity of ecosystem of pine wood nematode and its associated bacteria. [Methods] The metagenome of ecosystem of bacteria associated with the PWN was analyzed by 16S rRNA gene library and 454 sequencing. [Results] The results showed that 25 OTUs（Operational Taxonomic Units） were obtained from the library according to sequences similarity of 97%, which affiliated to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Bacteroidetes. The dominant bacteria were belonged to Gammaproteobacteria, especially Stenotrophomonas maltophilia in this class dominated the library. In terms of dominant bacteria, the results revealed by metagenome were similar to that of 16S rRNA gene library. [Conclusion] The diversity of bacteria associated with the PWN is high and these bacteria maybe have ecological role to the PWN.

Abstract:Abstract: [Objective] To isolate, identify and characterize a fungus with flocculating activity. [Methods] We used gradient dilution, plaque distribution and 18S rDNA analysis to isolate and identify a fungus with flocculating activity. We used high-speed centrifugation, ultrasonication and qualitative test to determine the nature of flocculating active substances. [Results] We isolated a flocculating active fungi and identified it as Penicillium purpurogenum. Ultrasonication test confirmed that the flocculating activity was primarily in the fermentation supernatant. We explored the flocculating activity curve and found that 4 days was the optimum fermentation time for accumulating flocculating active substances. The strain flocculating activity remains unchanged when pH varied from 2 to 11 and temperature varied from -70℃ to 100℃.We finally identified the flocculating active substances as saccharides. [Conclusion] We isolated a flocculating active strain P. purpurogenum EL-02, and identified its flocculating active products as saccharides.

Abstract:Abstract: [Objective] To understand the microbial community in mangrove sediments. [Methods] Sixteen stations were established in the Fugong mangrove area of Jiulong River Estuary, Fujian, China. Microbial community structure in this area was evaluated by PCR-DGGE method. The genetic diversity of microorganisms was analyzed based on the DGGE fingerprint. [Results] There were significant differences among the Shannon-wiener index, Richness and evenness. The possible reason might due to distinct location of samples from different stations. Bacterial diversity was higher in the mangrove areas than that in non-mangrove areas. Similarity analysis of bacterial communities in different station showed certain trends in similarity coefficients, and bacterial community structure similarity of the same transect was much higher than others. According to the results of sequence analysis of DGGE dominant bands, all of them were phylogenetically close to Proteobacteria, Acidobacteria and Chlorobia which belonged to uncultured microbes from coastal sediments in the estuary. [Conclusion] There are abundant microbial diversity and a large number of unknown microbial resources in mangrove sediments which need further research.

Abstract:Abstract：[Objective] To express and purify the Pro–Pro–Glu (PPE) family protein Rv1168c of Mycobacterium tuberculosis in E. coli. and to study the structure of Rv1168c. [Methods] The Rv1168c gene was amplified by PCR from Mycobacterium tuberculosis H37Rv strain genomic DNA and cloned into a prokaryotic expression vector pET32a The resulting recombinant expression plasmid pET32a-Rv1168c was then transformed into the E .coli strain DH5α and a high-level expression E. coli BL21(DE3) was established after induction with Isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE and mass spectrum analysis determined the relative molecular weight of this recombinant Rv1168c protein. It’s secondary and 3D structures were determined by circular dichroism and homologous modeling. [Results] The Mycobacterium tuberculosis Rv1168c gene (971bp) and high purified recombinant Rv1168c protein was obtained. The relative molecular weight of recombinant Rv1168c protein was determined to be 51.5kDa (vector included). Secondary structure of Rv1168c had about 34.4% α helix, 33.7%, β tune, 31.9% random coil at 25℃. Homologous modeling shows Rv1168c as （β/α）5 protein. [Conclusion] This study obtained purified recombinant Rv1168c protein and laid the foundation for exploration of the relationship between the structure and function of Rv1168c in the tuberculosis.

Abstract:Abstract：[Obiective] To understand whether hepatitis E virus (HEV) was infectious in sheep in Xinjiang . [Methods] We used reverse transcription-nested polymerase chain reaction(RT-nPCR) to detect HEVRNA in feces. The feces were collected from sheep with positive anti-HEV antibodies in a sheep farm in Xinjiang Autonomous Region. [Results] Six of 54 (11.11%) sheep were positive for HEV RNA. PCR amplification products were cloned, sequenced and analyzed. The homology among HEV of the 6 sheep HEV ORF2 189bp nucleotide amplification sequences was 99.38%-100%. They should belong to the same genotype. They were respectively compared with HEV genotypeⅠ, Ⅱ, Ⅲ and Ⅳ corresponding 189bp nucleotide sequences. The average homology was 78.67%-85.33%, 81.33%-82.67%, 78.67%-84.00% and 84.67%-95.36%. The maximum homology between 6 nucleotide amplification sequences and one sequence of genotype Ⅳ was 94.04%-95.36%. Based on sequence of the nucleic acid fragments, a phylogenetic tree was constructed. Six sheep HEV ORF2 189bp nucleotide amplification sequences in this study and bovine HEV, swine HEV and human HEV locate the same evolutionary vine and belong to genotype Ⅳ. [Conclusion] The finding suggested that infection of HEV probably exists in the sheep group in Xinjiang Autonomous Region and the sheep may be a new animal host except swine in origins of HEV infection.

Abstract:Abstract:[Objective] To evaluate the immuno-protective effect of GX0101Δmeq-BAC containing an infectious meq-null Marek’s disease virus genome. [Method] One-day-old SPF birds were reared separately in isolators with positive filtered air. On day 1 of age, chickens immunized with 10μg of GX0101Δmeq-BAC suspended in PBS, challenge infection with 500PFU very virulent rMD5 was performed at day 5 and 12 post-immunization separately. During 90 days after challenge, all bird were recorded and checked for necropsy. The samples of heart and liver were collected for histo-sections. [Results] The protective index of the two vaccines used was 87 and 33 for CVI988/Rispens and GX0101Δmeq-BAC, respectively, after challenged with the very virulent virus rMd5 at day 5 post-immunization. When challenged with rMd5 at day 12 post-immunization, the protection index of GX0101Δmeq-BAC increased to 53%. [Conclusion] Except that GX0101Δmeq-BAC can confer protection against very virulent Marek’s disease virus, a delay in the development of Marek’s disease could be observed in some chickens vaccinated with GX0101Δmeq-BAC. On the other hand, compared with CVI988/Rispens, the reconstruction of GX0101Δmeq-BAC in the body is a prerequisite for access to protection. Therefore, there is a blank period after immunization, which provides a chance for infection with the wild Marek’s disease virus.

Abstract:Abstract: [Objective] To explore whether polyethylene glycol (PEG) as adjuvant could enhance the humoral and cellular immune responses on hepatitis B virus DNA vaccine. [Methods] C57BL/6J mice were immunized with PEG and pVAX-S2 or alone pVAX-S2. After these mice were finally immunized for 14 days, the anti-HBs IgG, T cell proliferation, the expression of cytokines and CTL in vivo were detected. [Results] Compared to mice immunized with alone pVAX-S2, PEG as adjuvant could increase the production of anti-HBs IgG and HBsAg specific T cell proliferation. In addition, the expression of IL-4, IFN-γ in CD4+ T cells and IFN-γ in CD8+ T cells was higher than control groups. The PEG/pVAX-S2 groups could elicit significantly in vivo HBsAg specific CTL responses. [Conclusions] PEG as adjuvant could enhance humoral and cellular immune responses, as well as in vivo CTL activity.

Abstract:Abstact:[Objective] Effect of various culture conditions on the morphology, amount and carbonic anhydrase (CA) activity of Bacillus mucilaginosus were examined, as well as the effect on calcium carbonate crystal forming, shape and amount. [Methods] The strain was inoculated in N-free or N-containing medium, and the bacterial morphology, number and CA activity were compared under different culture conditions. By collecting different cultures and adding them to the system of calcium carbonate crystallization we studied the relationship between the bacteria and the formation of calcium carbonate crystals. [Results] A small number of cell, capsular hypertrophy, lower CA activity in bacterial culture were obtained under N-free culture condition. In contrast, more biomass quantity, thin capsule, and high CA activity were got in the nitrogen-containing culture. In the calcium carbonate crystal system, adding N-free culture of bacteria produced a smooth surface of calcium carbonate crystals, larger volume but small density, the addition N-containing culture of bacteria formed rough surface, bigger density but smaller volume of calcium carbonate crystals. [Conclusion] Different culture conditions can cause significant differences in bacterial amounts, capsular thickness and CA activity, and then influence the crystal growth and form of calcium carbonate.

Abstract:Abstract: [Objective] We cloned PsNCS1 encoding neuronal calcium sensor from Pst and analyzed its transcriptional profile. [Methods] A full-length cDNA of PsNCS1 was cloned by using RT-PCR in combination with cDNA library screening, the sequence was analyzed with different bioinformatic tools and the gene expression pattern was characterized via real-time RT-PCR. [Results] PsNCS1 (Genbank accession no. GU134621) encoded 190 amino acids, with a molecular weight of 22.17 KDa and a pI of 4.96. PsNCS1 contained four conserved EF-hand domains and was N-terminally myristoylated. Phylogenetic analysis indicated that PsNCS1 was highly similar to the NCS from Basidiomycetes and the highest similarity was with that from Puccinia graminis (96%). Real-time RT-PCR analysis indicated the amount of PsNCS1 transcripts of urediospores and of germinated urediospores were doubled or more comparing with those of fungal bodies at other different developmental stages. [Conclusion] PsNCS1 might be involved in the process of urediospore formation and germ tube elongation. The present results may provide basic data for further analysis of the role of PsCNS1 in pathogenesis process and calcium signaling.

Abstract:Abstract: [Objective] To implement inducible and constitutive over-expression of Yarrowia lipolytica lipase gene lip1 in Pichia pastoris using codon optimization. [Methods] We cloned Y. lipolytica lipase gene lip1 according to codon bias of P. pastoris, and optimized lip1 using overlap extension PCR synthesis. Then, we cloned the original and optimized genes into the induced vector pPIC9K and newly built constitutive carrier pGAP9K, and electrotransformated the resultant expression plasmids into P. pastoris GS115. Through G418 resistance screening, high copy transformatants were selected and fermented in shake flasks. P-nitrophenyl palmitate (pNPP) was used as substrates for assay the activities of the expressed lipase, and the characteristics of the lipase were further examined. [Results] We successfully cloned lipase gene lip1 from Y. lipolytica, nucleotide sequence revealed that the open reading frame (ORF) had 1461 nucleotides, encoding 486 amino acids, without any intron or any signal peptide. SDS-PAGE analysis and fermentation result showed that the optimized gene had a higher expression level than the original one, and the constitutive expression was superior to the inducible expression. Preliminary analysis showed that the optimal substrate of Lip1 was p-nitrophenyl butyrate (C4), the optimum temperature and pH was 45℃and 8.5, respectively. [Conclusion] Y. lipolytica lipase gene lip1 can be over-expressed through both inducible and constitutive expressions using codon optimization, which lays a solid foundation to further study Y. lipolytica lipase family, and also provides an important prerequisite for scale production and industrial application of the lipase.