Abstract:To better adapt to the environment, prokaryocyte can take up exogenous genes(from bacteriophages, plasmids or genomes of other species) through horizontal gene transfer. Accompanied by the acquisition of exogenous genes, prokaryocyte is challenged by the invasion of 'selfish genes'. Therefore, to protect against the risk of gene transfer, prokaryocyte needs to establish mechanisms for selectively taking up or degrading exogenous DNA. In recent years, researchers discovered an adaptive immunity, which is mediated by the small RNA guided DNA degradation, prevents the invasion of exogenous genes in prokaryocyte. During the immune process, partial DNA fragments are firstly integrated to the clustered regularly interspaced short palindromic repeats(CRISPR) located within the genome DNA, and then the mature CRISPR RNA transcript and the CRISPR associated proteins(Cas) form a complex CRISPR/Cas for degrading exogenous DNA. In this review, we will first briefly describe the CRISPR/Cas systems and then mainly focus on the recent advances of the function mechanism and the regulation mechanism of the type I-E CRISPR/Cas system in Escherichia coli.

Abstract:Anaerobic ammonia oxidation(Anammox) is one of the highlights in the microbiology and environmental research. Anammox is the metabolic function of anaerobic ammonium oxidation bacteria(AnAOB). Different from aerobic ammonia oxidizing bacteria(AAOB), AnAOB has its characteristics in phylogenetics, cytology, physiology, etc. Especially, it has the unique organelle called anammoxosome. Anammoxosome is the place where the metabolism takes place. The research of anammoxosome is the basis to figure out the anammox performance. This article discusses the composition, structure and function of the anammoxosome.

Abstract:Epstein-Barr virus(EBV) is the first identified human oncogenic DNA virus in the gamma-herpesvirus family. EBV triggers a cascade events of innate immune responses through Toll-like receptor signaling including the production of type I interferons and the activation of functional autophagy. However, EBV has developed much more elaborate and sophisticated strategies for subverting and escaping the host immune system, such as limiting its own gene expression, activing the host ubiquitin-specific protease system, and interfering ubiquitin modification. EBV impairs the host immune system, leading to lifelong persistent infections, which in turn result in the occurrence of EBV-associated diseases, such as nasopharyngeal carcinoma and infectious mononucleosis. Thus, to better understand the mechanisms regarding the infection latentency and oncogenicity of EBV invasion will be crucial for identifying potential immunotherapeutic targets for EBVrelated diseases, such as infectious mononucleosis and nasopharyngeal carcinoma. In this article, we discuss the research advances regarding the virology and immunology of EBV in the modulation of the host immune response and evasion.

Abstract:Arbuscular mycorrhizal(AM) fungi play an important role in energy flow and nutrient cycling, besides their wide distribution in the cosystem. With a long co-evolution, AM fungi and host plant have formed a symbiotic relationship, and fungal lipid metabolism may be the key point to find the symbiotic mechanism in arbusculart mycorrhiza. Here, we reviewed the most recent progress on the interaction between AM fungal lipid metabolism and symbiotic signaling networks, especially the response of AM fungal lipid metabolism to symbiotic signals. Furthermore, we discussed the response of AM fungal lipid storage and release to symbiotic or non-symbiotic status, and the correlation between fungal lipid metabolism and nutrient transfer in mycorrhiza. In addition, we explored the feedback of the lipolysis process to molecular signals during the establishment of symbiosis, and the corresponding material conversion and energy metabolism besides the crosstalk of fungal lipid metabolism and signaling networks. This review will help understand symbiotic mechanism of arbuscular mycorrhiza fungi and further application in ecosystem.

Abstract:[Objective] Low pathogenic avian influenza(LPAI) H9N2 subtype virus has been prevalent in domestic poultry in China over two decades. This study was to determine the genetic evolution trend of H9N2 avian influenza virus(AIV) under immune pressure of vaccine.[Methods] H9 HA sequences of 40 isolates from the present study and 136 pandemic strains and 7 classical strains from China downloaded from GenBank, were genetically analyzed to determine evolution, molecular characteristic, and mutation frequency.[Results] Phylogenetic trees analysis suggested that H9N2 subtypes AIV could be clustered into 5 distinct lineages:G1-like, BJ94-like, Y280-like, S2-like and Americans lineages. Most H9N2 isolates in 2005-2014 belonged to S2-like sub-genotype, suggesting that this genotype was the dominate isolates in China. Further more, comparison based on the amino acid sequence showed that different lineages have their distinct characteristics, and significant accumulations of amino acid variation were also found. In addition, in comparison with reference Ck/BJ/1/1994 HA gene, average annual substitution rates of H9N2 pandemic strain nucleotide and amino acid were 5.73×10^-3 and 4.25×10^-3 from 1994 to 2014, respectively. Substitution rate during 2011-2014 were 6.35×10^-3 and 5.32×10^-3, higher than that during the period of 2006-2010(5.22×10^-3 and 3.70×10^-3) and even much higher than that during the 1999-2005(0.74×10^-3and 0.50×10^-3), when the vaccines were initially applied in the field.[Conclusion] Overall, these data indicate that the mismatch between H9N2 vaccine strains and pandemic strains drives the virus to quickly mutate.

Abstract:[Objective] We screened bacteria producing L-aspartate α-decarboxylase from grapery soil and optimized the fermentation conditions.[Methods] L-aspartate α-decarboxylase producing bacteria were screened by color-changing circle and liquid secondary screening culture media. Combination of morphological, physiological and biochemical characteristics and 16S rRNA sequence analysis were used to identify the bacteria. Fermentation conditions were optimized by single factor test and orthogonal experiment.[Results] Strain PanD37 showed high L-aspartate α-decarboxylase producing property and was identified as Bacillus tequilensis. The optimum fermentation conditions of PanD37 were liquid volume of 50 mL in 500 mL flask, 220 r/min at 35℃, inoculation amount of 5% for 28 h with a medium of 22.5 g/L sucrose, 7.5 g/L fumaric acid, 20 g/L peptone, 6 g/L L-aspartic acid, 2 g/L Triton X-100, at initial pH of 7.0. Under the optimal fermentation conditions, the highest L-aspartate α-decarboxylase activity reached 44.57 U/mL, which was 2.57 folds higher than that obtained before optimization.[Conclusion] Strain PanD37 was identified as Bacillus tequilensiswhich was capable of highly producing L-aspartate α-decarboxylase under the optimal fermentation conditions.

Abstract:[Objective] We studied several crucial factors influencing the uridine biosynthesis in Bacillus subtilis, including mutations of phosphoribosylpyrophosphate synthetase(PRPP synthetase)(prs) and carbamyl phosphate synthetase(pyrAA/pyrAB), and overexpression of heterologous 5'-nucleotidase(sdt1).[Methods] According to the inferred allosteric sites, we introduced point mutation into coding sequences of prs and pyrAB. The mutated prs gene was integratedly expressed in the xylR locus of the chromosome and the pyrAB gene was modified in-situ. The sdt1 gene was overexpressed in the saB locus of the chromosome. The effect of the genetic modification on uridine biosynthesis was characterized by the analysis of uridine, cytidine and uracil in the fermentation broth.[Results] The mutations of Asn120Ser, Leu135Ile, Glu52Gly or Val312Ala on PRPP synthase resulted in an increase of uridine production by 67% and 96%, respectively. The mutations of Ser948Phe, Thr977Ala and Lys993Ile on carbamyl phosphate synthase resulted in a 182% increase of uridine yield to 6.97 g/L. The overexpression of heterologous 5'-nucleotidase resulted in a 17% increase of uridine yield to 8.16 g/L.[Conclusion] The activity and regulation mechanism of PRPP synthase and carbamyl phosphate synthase was an important factor to limit the excessive synthesis of uridine. Asn120Ser and Leu135Ile mutations of PRPP synthase and Ser948Phe, Thr977Ala and Lys993Ile mutations of carbamyl phosphate synthase will facilitate the biosynthesis of uridine. The additional Glu52Gly and Val312Ala mutations of PRPP synthase were beneficial for uridine biosynthesis. The reaction from UMP to uridine also limited the biosynthesis of uridine in B. subtilis.

Abstract:[Objective] To study the active sites of N-acetylhexosamine 1-kinase(NahK) from Bifidobacterium longum JCM1217.[Methods] We obtained expression strains of 10 single-mutants at 4 sites of NahK by site-directed mutagenesis, and expressed and purified both wild-type(WT) and mutant enzymes. Then, their optimum pH and optimum concentration of Mg²⁺ were determined by DNS assay and NADH-coupled microplate photometric assay, and their kinetic parameters were measured.[Results] Four mutants(D208A, D208N, D208E and I24A) lost most part of the catalytic activity. The optimum pH of mutants H31A, H31V, F247A and I24V switched from pH 7.5(for WT) to pH 7.0, and the optimum concentration of Mg²⁺ of mutants H31A and F247A increased to 10 mmol/L from 5 mmol/L(for WT). The kinetic parameters of WT and mutants indicate that mutant F247Y had higher enzymatic activity toward GlcNAc, GalNAc and ATP than WT.[Conclusion] The key amino acids that affect the catalytic activity of NahK were determined by site-directed mutagenesis, and together with the mutant that has higher catalytic efficiency, this has laid a foundation for further modification and evolution of NahK.

Abstract:[Objective] In this study, a new chimeric protein SEG expressed in previous work was applied to evaluate its translocating efficiency of shRNA to rabies virus infected cells in mice, meanwhile, the capability of anti-rabies virus was investigated.[Methods] Rabies virus strain CVS-24 was inoculated into the hind leg to establish a mouse model of rabies in a dose of 50 LD₅₀; 12 h thereafter the mice were injected intravenously with shRNA-producing plasmid mixed with SEG. To test shRNA delivery, single-cell suspensions from brain, spleen and liver were examined by flow cytometry. Rabies virus in brain tissue of mice was detected by qRT-PCR, RT-PCR, western blot and directed immunofluorescence assay. Mice were monitored for survival and serum samples were tested for IFN-α levels.[Results] No green fluorescent protein(GFP) was seen in the spleen or liver, suggesting that SEG allows specific targeting of RV-infected cells. RT-PCR and western blot showed that mice treated with SEG-shRNA had lower rabies virus RNA and protein levels than the controls. Real-time PCR showed that rabies virus was reduced 4.88 fold compared to the mock cells. Survival of RV-infected mouse showed a significant protection from rabies virus infection by SEG-shRNA treatment. The survival was up to 50% whereas the control group all died. IFN was not induced in SEG-shRNA treated animals.[Conclusion] shRNA-producing plasmid was specifically delivered into rabies virus infected cells using the SEG protein, and effectively inhibited rabies virus gene expression and replication in vivo. SEG-shRNA can be used for adjuvant treatment for rabies.

Abstract:[Objective] To investigate the diversity of culturable sulfur-oxidizing bacteria in hydrothermal vent environments of the South Atlantic, and analyze their characteristics of sulfur oxidation.[Methods] We enriched and isolated sulfur-oxidizing bacteria from hydrothermal vent samples collected from the South Atlantic. The microbial diversity in enrichment cultures was analyzed using the Denatural Gradient Gel Electrophoresis method. Sulfur-oxidizing characteristics of the isolates was further studied by using ion chromatography.[Results] A total of 48 isolates were obtained from the deep-sea hydrothermal vent samples, which belonged to 23 genera and mainly grouped into alphaProteobacteria(58.3%), Actinobacteria(22.9%) and gama-Proteobacteria(18.8%). Among them, the genus Thalassospira, Martelella and Microbacterium were dominant. About 60% of the isolates exibited sulfur-oxidizing ability and strain L6M1-5 had a higher sulfur oxidation rate by comparison analysis.[Conclusion] The diversity of sulfur-oxidizing bacteria in hydrothermal environments of the South Atlantic was reported for the first time based on culture-dependent methods. The result will help understand the biogechemical process of sulfur compounds in the deep-sea hydrothermal environments.

Abstract:[Objective] X-ray micro-computed tomography(micro-CT) technology, as used in the in situ and nondestructive analysis of soil physical structure, provides the opportunity of associating soil physical and biological assays. Due to the high heterogeneity of the soil matrix, X-ray micro-CT scanning and soil microbial assays should be conducted on the same soil sample. This raises the question whether X-ray micro-CT influences microbial function and diversity of the sample soil to be analyzed.[Methods] To address this question, we used plate counting, microcalorimetry and pyrosequencing approaches to evaluate the effect of X-ray-at doses typically used in micro-CT-on soil microorganisms in a typical soil of North China Plain, Fluvo-aquic soil and in a typical soil of subtropical China, Ultisol soil, respectively.[Results] In both soils radiation decreased the number of viable soil bacteria and disturbed their thermogenic profiles. At DNA level, pyrosequencing revealed that alpha diversities of two soils biota were influenced in opposite ways, while beta diversity was not affected although the relative abundances of some guilds were changed.[Conclusion] These findings indicate that the metabolically active aspects of soil biota are not compatible with X-ray micro-CT; while the beta molecular diversity based on pyrosequencing could be compatible.

Abstract:[Objective] From the previous comparative genomic analysis, we found a specific unknown 10 kb sequence(including 11 Open reading Frames) in Chinese piscine strain GD201008-001 genome. To study the role of 10 kb in the pathogenicity of piscine S. agalactiae, the 10 kb sequence was deleted from the GD201008-001 genome.[Methods] The isogenic mutant Δ10 kb was constructed by using the temperature-sensitive Streptococcus-E. coli shuttle vector pSET4s. We compared the growth characteristics, adherence to HEp-2 cell and bacterial virulence in a zebrafish infection model between wild strain and mutant. Meanwhile the expressions of the known virulence genes from GD201008-001 and Δ10 kb were also quantified by real-time PCR.[Results] The Δ10 kb showed no significant differences in bacterial morphology and adherence to HEp-2 cells compared with the wild-type strain, but the speed of growth was slightly slower than the wild strain. Furthermore the 50% lethal dose of Δ10 kb was decreased up to 10-fold(P<0.001) of the parental strain in a zebrafish infection model, and the expressions of the virulence genes, PI-2b and neul, were significantly increased in the mutant.[Conclusion] These findings demonstrated that the 10 kb sequence of piscine Streptococcus agalactiae exerts a significant effect on bacterial virulence and probably regulates the virulence genes expression of GD201008-001.

Abstract:[Objective] To develop a bivalent vaccine against pseudorabies virus(PRV) and porcine circovirus(PCV2), IL-18 was used as immunologic adjuvant.[Methods] Porcine IL-18 gene was inserted into vector pGO. The obtained recombinant transfer plasmid pGO18 was transfected into ST cells with PRV attenuated vaccine HB98 strain. Then plaque selection and purification were performed to obtain purified recombinant virus PGO18. RT-PCR and Western blot were used to demonstrate the expression of PGO18 from transcription and protein levels, respectively. Six-week-old female Kunming mice were immunized with recombinant virus PGO18 and PGO, commercial PCV2 inactivated vaccine, PRV attenuated vaccine HB98 strain, 1640 medium. Mice were vaccinated twice 4 weeks later and then challenged with the virulent PCV2 DF strain and PRV Min/A strain 4 weeks after the second immunization. ELISA, serum neutralization assay, flow cytometry and protect experiment were used to demonstrate the immunity of mice.[Results] The recombinant virus PGO18 was obtained, and it could express on ST cells. Mice vaccinated with PGO18 elicited high levels of humoral and cell immune response, and could also be protected against PCV2 and PRV challenge.[Conclusion] The recombinant virus possessed high safety and good immunogenicity. It may be a candidate vaccine strain against PCV2 and PRV infection.

Abstract:[Objectives] High-throughput sequencing technology is increasingly applied in intestinal microbiota of aquatic animals including shrimp. However, there is a lack of standard method or kit for DNA isolation from shrimp intestinal microbiota, and little is known about the effectiveness and biases regarding DNA extraction based on high-throughput sequencing. The aim of this study was to study the biases of different DNA extraction kits on community structure of shrimp intestinal microbiota through high-throughput sequencing, and to better understand the structure and composition of bacterial flora associated with healthy Litopenaeus vannamei.[Methods] We extracted the total DNA of intestinal microbiota from L. vannamei with three commercial kits designed for DNA extraction from bacteria, stool and tissue(Omega, USA). DNA quality was evaluated based on the absorbance ratios of 260/280 nm by NanoDrop, while DNA concentration was quantified using PicoGreen. Then Illumina MiSeq high-throughput sequencing was used to examine the intestinal bacterial communities following PCR amplification of 16S rDNA V4 region.[Results] The yield and purity of the DNA from the Bacterial Kit(SIB) were superior to those from the Stool Kit(SIS), whereas the DNA from Tissue Kit(SIT) presented too small amount to be amplified efficiently. The average sequence reads obtained from SIB and SIS samples were 52151±5085 and 55296±5147 respectively. After resampling at the same depth of 46800 reads, the operational taxonomic unit(OTU) number and Shannon diversity index of SIS samples were significantly higher than those of SIB samples. By contrast, the reproducibility of OTU among SIB replicates was higher than that among SIS replicates. The dominant phyla of SIS and SIB samples were identical, including Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, Actinobacteria, and Cyanobacteria. However, the relative abundances of almost all the dominant groups at various taxonomic levels differed greatly between these two samples.[Conclusion] Significant biases on community structure of shrimp intestinal microbiota were detected which originated from DNA extraction. And the core microbiota of the healthy L. vannamei in this study was mainly composed of genera Photobacterium, Lactococcus, Aliivibrio, Vibrio, as well as three other unclassified groups.

Abstract:[Objective] To study the transcriptional regulation of the structural components Hcp1 by H-NS in Vibrio parahaemolyticus.[Methods] Expression of Hcp1 in the wide-type(WT) strain and hns mutant(Δhns) were detected by Western blot using rabbit anti-Hcp1 polyclonal antibodies. Total RNAs were extracted from WT and Δhns strains. Quantitative RT-PCR was carried out to calculate the transcriptional variation of hcp1 between WT and Δhns strains, and then primer extension assay was used to detect the transcription start site and the promoter activity(the amount of primer extension product) of hcp1 in WT and Δhns. The entire promoter region of hcp1 was amplified by PCR with ExTaq^TM DNA polymerase using WT genomic DNA as the template. The over-expressed His-H-NS was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns(Amersham). Electrophoretic mobility shift assay(EMSA) was applied to analyze the DNA-binding activity of His-H-NS to hcp1 promoter region in vitro.[Results] Western blot and quantitative RT-PCR results showed that expression of Hcp1 was inhibited by H-NS. The primer extension assay detected only one transcription start site located at 62 bp upstream of hcp1, whose transcription was H-NS and σ⁵⁴-dependent. EMSA result indicated that His-H-NS was able to bind the promoter DNA region of hcp1.[Conclusion] The expression of hcp1 was directly repressed by H-NS in V. parahaemolyticus.

Abstract:[Objective] To study the epidemiological and molecular characteristics of Campylobacter jejuni in poultry in Hubei province, we used multilocus sequence typing method to classify 47 local C. jejuni strains.[Methods] Genomic DNA of each isolated strain was extract, seven housekeeping genes including aspA, glnA, gltA, glyA, pgm, tkt and uncA were amplified by PCR and sequenced, and then the sequences of genes were analyzed using MLST database.[Results] There were a total of 38 sequence types and 10 clonal complexes, and ST353 and ST464 complexes were the largest amount of the population of C. jejuni analyzed, of which 2 new allelic profile and 25 new sequence types were found. Phylogenetic tree shows that sequence types from different types of poultry and different regions were different.[Conclusion] Forty-seven C. jejuni strains isolated from poultry in Hubei were analyzed using MLST and showed abundant genetic diversity, it will provide scientific data to the epidemiological investigation of C. jejuni in Hubei, China.