Abstract:[Objective] The aim of this study was to study the influence of Crc (Cataboliterepression control protein) on pyoluteorin (Plt) biosynthesis and gene expression in Pseudomonas protegens H78. [Methods] The crc gene in the H78 chromosome was knocked out by homologous recombination. Plt production of H78 strain and its mutant grown in KMB was measured by HPLC. The regulation of Crc on plt operon expression was assessed by the lacZ reporter analysis. [Results] Plt production was significantly reduced in H78Δcrc. Crc positively regulated the expression of plt biosynthetic operon at the overall level, the transcriptional level and the post-transcriptional level. [Conclusion] The global regulator Crc positively regulates Plt production and its operon expression.

Abstract:[Objective] To study the effect of Crp family transcriptional regulator gene fnrN mutation on the fermentation characteristics and genes expression in Agrobacterium sp. ATCC 31749. [Methods] A suicide plasmid pJQ-fnrN-kan was constructed and transformed into wild type strain by triparental conjugation method to obtain the fnrN mutation strain (ΔfnrN). The cell growth and fermentation characteristics of ΔfnrN were compared with that of the wild strain, then comparative transcriptomes with RNA-Seq were used to analyze the expression changes of genes in curdlan-producing phase due to fnrN mutation. [Results] The level of curdlan production was reduced by 22.0% because of fnrN mutation. Transcriptomes analysis revealed that 186 genes expressed significantly different (|log2(|fold change|)|≥1 and q≤0.001), and about 65% differently expressed genes were up-regulated. The key genes (crdASC) in curdlan synthesis were repressed in different degree, the fnrN mutation significantly down-regulated the expression of ecfR and sinR, coding ECF family RNA polymerase sigma factor and Fnr family regulator of biofilm formation respectively. The transcription levels of genes (cydAB, cy2 and fixNOPQ) related with cytochrome were down-regulated 2?13 times. [Conclusion] The fnrN regulates curdlan synthesis by controlling the expression of ecfR and sinR, and takes part in oxygen signal regulation through down-regulating the expression of fixNOPQ, which is helpful to enrich the understanding of oxygen regulation system in Agrobacterium sp.

Abstract:[Objective] To analyze the regularity of the activity of respiratory chains and the transcriptional levels of related genes in Acetobacter pasteurianus under the stress of high acetic acid concentration, A. pasteurianus CICC 20001 and A. pasteurianus ATCC 33445 were studied. [Methods] The biomass, acid production and enzyme activity of the two strains have been analyzed in different initial acetic acid concentrations of 0, 1%, 2% and 3%. Real-time quantitative PCR was used to analyzed changes of relative transcriptional levels of respiratory chains related enzyme synthesis genes in different concentrations. [Results] It was found that acid production capacity of two strains was the strongest when initial acetic acid concentration was 1%; and their average acid production rate reached 0.667 g/(L·h) during 48 hours fermentation. At the same time, enzyme activity levels of ADH (alcohol dehydrogenase) and ALDH (acetaldehyde dehydrogenase) also rise to the highest of 12.01 and 9.77 U/mg on average. And the relative transcriptional level of the respiratory chain related enzyme synthesis genes were also increased compared with initial acid of 0. The enzyme activity and acid production capacity decreased gradually when initial acetic acid concentration increased to 2% and 3%. The adh, cyt bd, cyt o and fapA gene on respiratory chain maintained a relatively high transcriptional level while the transcriptional level of rest genes decreased. [Conclusion] It is identified that under high-intensity acetic acid conditions, the transcriptional level of adh gene is increased spontaneously, which activates the substrate phosphorylation and pump acetic acid out. At the same time, the aldh gene’s expression is inhibited, which causing the reducing of acid production and maintaining rather low concentration of acetic acid. In addition, the level of other efflux enzymes in the respiratory chain will also increase, such as cyt bd and cyt o, which enhance the substrate phosphorylation in metabolic process and accelerate the release of energy.

Abstract:[Objective] In order to research the characteristics of artificially constructing algae symbiosis system in oil production. [Methods] aseptic Chlorella was screened and isolated from the BG11 medium, the growth and oil-producing characteristics of Chlorella in the symbiosis system were studied by artificial construction of co-culture system of algae and bacteria. [Results] The results showed that the growth of algae, accumulation of oil and the production of fatty acid components of biodiesel were significantly higher in the co-culture system of algae and bacteria compare to that of aseptic Chlorella. Co-culture system of bacteria (Stenotrophomonas maltophilia) and Chlorella has the best effect, and the biomass, oil content and C18-1 of Chlorella increased by 9%, 36.3% and 259.2% respectively. [Conclusion] This study shows that the quality of microalgae biodiesel with good utilization value can be improved by artificial construction of co-culture system of algae and bacteria.

Abstract:[Objective] Inositol, also named cyclohexanol, is a kind of bioactive sugar alcohol. It was widely used in pharmaceutical, food and feed industries. To obtain the microbial cell factory that produced more inositol, the recombinant Saccharomyces cerevisiae was constructed by metabolic engineering. [Methods] Positive and negative regulation of inositol biosynthesis was modified. The Inositol-3-phosphate synthase gene (ino1) was overexpressed in S. cerevisiae. Meanwhile, the transcription inhibitor gene (opi1) that inhibits the biosynthesis of inositol and the antibiotic resistance gene (kanMX) for screening strains were knocked out of recombinant S. cerevisiae. Inositol content in fermentation broth of engineered strains was detected by Gas chromatography. [Results] Biological safety engineered strain S. cerevisiae was constructed and shake flask fermentation results showed the yield of inositol was 1.021 g/L. [Conclusion] Over-expression of ino1 gene and opi1 gene knocked out could efficiently enhance the productivity of inositol in S. cerevisiae. The results provided the foundation for industrial application of inositol production by microbial fermentation.

Abstract:[Objective] We designed and did experiments to simulate canopy damage in Xiaokeng forest farm, and to analyze the impact of canopy openness and litter input on soil CO2 fixation bacteria community structure. [Methods] A manipulative field experiment was conducted to study the effects of four different forms: control plots, damage-removal and treatment, damage-retention and processing, and untreated-add foliage treatment. Soil CO2 fixation bacteria community structure was measured by MiSeq analyses after a year of canopy damage. [Results] Based on bioinformatics and statistical analyses, community richness of soil CO2 fixation bacteria decreased and diversity increased under the influence of litter inputs and canopy openness. Nitrosospira increased significantly and became the dominant population, whereas the original dominant group of Bradyrhizobium decreased. Principal component analysis shows that community structures of soil CO2 fixation bacteria were changed. [Conclusion] A large number of litter inputs and increased canopy openness improved diversity of soil CO2 fixation bacteria, but decreased their community richness, and affected their community structure.

Abstract:[Objective] Investigated the distribution and species of Trichoderma in Dongting Lake wetland, Hunan province, supplemented Trichoderma resources of China, and farther more use of functional strains screening and applied. [Methods] Species were identified by analysis of internal transcribed spacer regions of the rRNA gene cluster (ITS) and morphological characteristics, and analyzed the genetic relationship based on ITS sequences phylogenetic tree. The antifungal activity were determined by mycelial growth rate method, and hydrolase activity were determined by hydrolyzation radius, screening the superior comprehensive biocontrol Trichoderma strains by gray correlative degree analysis. [Results] Total 114 Trichoderma strains were isolated from 52 soil samples and 18 water samples, 15 known species were identified as followed: T. harzianum, T. virens, T. asperellum, T. saturnisporum, T. hamatum, T. koningiopsis, T. brevicompactum, T. atroviride, T. erinaceum, T. capillare, T. longibrachiatum, T. ovalisporum, T. pleuroticola, T. ghanense, T. crassum, among them T. capillare was Chinese new record, besides Trichoderma sp. CK-2016 was an uncertain new species. T. harzianum was the dominant species with 19.30% quantity among them. All these Trichoderma species belonged to 7 Clade as followed: Harzianum Clade, Virens Clade, Longibrachiatum Clade, Lutea Clade, Viride Clade, Hamatum Clade, Unknown Clade. Gray correlative degree analysis showed that the grey relation degree of three strains TW21990, QT22040 and QT22094 were 0.849 5, 0.798 6 and 0.732 6, respectively, and were better comprehensive biocontrol Trichoderma strains among primary twenty-one strains. [Conclusion] The Trichoderma species in Dongting Lake wetland, Hunan province have diversity with a new China record of T. capillare and an uncertain new species among them, T. harzianum strain TW21990, T. longibrachiatum strain QT22040 and T. ovalisporum strain QT22094 are potential biocontrol agents.

Abstract:[Objective] To evaluate microbial biodegradation, three kinds of soil, swamp, orchard and farmland were selected. And to find dominant microbe that can degrade the PLA, we characterize the different of abundance, diversity and community structure of the three microbial strain. [Methods] The residual degraded PLA were analyzed by SEM, the tensile strength, elongation at break and CO2 generation analysis. And characterize the different of abundance, diversity and community structure of the three kinds of soil by using pyro-sequencing. [Results] The PLA biodegradation in swamp, orchard, and farmland were 13.7%, 10.6%, 4.5%, respectively. The taxonomic analysis of the pyro-sequencing data was grouped into 9 different phyla and 16 different class. [Conclusion] By comparing the microbial community of three kinds of soil, Proteobacteria and Bacteroidetes were the two most dominant phylato degrade the PLA. Flavobacteriaceae, Comamonadaceae and Cytophagaceae were the three most dominant class to degrade the PLA. The result provides scientific basis for the study of microbial diversity and community structure in biodegradation of PLA.

Abstract:[Objective] This study aimed to study the effect of different free chlorine concentration and exposure time on bacterial inactivation as well as cellular adenosine triphosphate (ATP). [Methods] Inactivation behaviors of autochthonous bacteria from river water were investigated with flow cytometry (FCM). Total cell counts (TCC), intact cell counts (ICC) and intracellular and extracellular (ATP) were analyzed during the chlorination process. [Results] The results showed that both chlorine concentration and exposure time influenced the removal efficiency of bacteria. At a low concentration of chlorine (<2 mg/L), a prolonged exposure time was needed to achieve a high removal efficiency of bacteria. In contrast, at a high concentration of chlorine (≥2 mg/L), inactivation of more than 90% of bacteria could be achieved within a short exposure time (i.e. 1 min). The results also indicated high nucleic acid (HNA) content bacteria were more vulnerable than low nucleic acid (LNA) content bacteria during the chlorination process. Furthermore, it was observed that concentration of intracellular ATP decreased with increasing chlorine doses. However, the increase of extracellular ATP was only recorded at high chlorine doses (≥2 mg/L). [Conclusion] Chlorination resulted in a decrease of ICC, and reduction of bacterial activity with increasing chlorine concentration. The results demonstrated that FCM and ATP measurements are useful and fast tools to assess inactivation kinetics of indigenous riverine bacteria during the chlorination treatment.

Abstract:[Objective] In this study, two L-aspartate α-decarboxylase genes (panD) from Listeria monocytogenes (panDL.m) and Corynebacterium jeikeium (panDC.j) were expressed in Escherichia coli, respectively, and the recombinant enzymes were purified and characterized. [Methods] The panD genes from L. monocytogenes and C. jeikeium were synthesized and inserted into pET28a(+) to obtain expression plasmids, which was then transformed into E. coli BL21(DE3). PanDL.m and PanDC.j were functionally expressed and the recombinant enzymes were purified by HisTrapTM affinity chromatography. Their catalytic properties were characterized and the effects of substrate concentration on enzymatic conversion were studied. [Results] Those α and β subunits were detected by Tricine-SDS-PAGE, indicating that both of the PanDs were processed by self-cleavage. The specific activities were 8.9 U/mg for PanDL.m and 11.8 U/mg for PanDC.j. They exhibited the same optimal temperature at 60 °C, while they possessed different optimal pH at 7.0 and 6.0, respectively. Both the enzymes were stable at the condition of 30?50 °C, and pH 4.0?7.0. Compared with the most studied PanD from C. glutamicum, the substrate inhibition effect of the PanDL.m was significantly less. [Conclusion] Under the high temperature and acidic conditions, PanDL.m as well as PanDC.j could specifically transform L-aspartic to β-alanine. PanDL.m could be more appropriate for industrial application because of less substrate inhibition.

Abstract:[Objective] To study the screening and application antagonisms of Fusarium graminearum in order to lay the foundation for use of bio-control agents for corn stalk rot. [Methods] The antagonism of F. graminearum were screened from corn endophytic bacteria using the confrontation culture method. A bacterial strain (named as 48SJ7-1) which showed inhibition to F. graminearum was obtained. The bacterial strain was identified according to its physiological and biochemical characteristics and a 16s rRNA gene sequence analysis; and its effect on corn stalk rot caused by F. graminearum was determined by pot experiment. [Results] Strain 48SJ7-1 was identified as Bacillus methylotrophicus (accession no. KU377993). In pot experiments, application of 48SJ7-1 reduced corn stalk rot by 68.47% compared to the non-treated control, and there was no statistically significant difference with the 2% tebuconazole FSC treated plants. [Conclusion] F. graminearum, the main pathogenic fungi of corn stalk rot, can be effectively controlled by 48SJ7-1. The potting results showed that 48SJ7-1 could promote maize growth and no phytotoxicity was observed.

Abstract:[Objective] We identified avirulence genes of rice blast fungus in Taojiang, Hunan Province, to deploy rice blast resistant varieties and blast resistant breeding in Hunan. [Methods] We collected samples of leaf blast of Lijiangxintuanheigu (LTH) in Taojiang and isolated blast fungi through single spore isolation. Avirulence genes of these fungi were identified by in vitro after being inoculated onto the 5th leaf of the 24 monogenic near-isogenic lines against blast at 5 leaves stage which is cultivated from LTH with known blast-resistance gene. We analyzed the interactions between the blast-resistance genes combining pathogenicity association coefficient (PCA) with resistance association coefficient (RAC). [Results] The total 92 strains of M. oryzae contain the whole 24 avirulent genes based on 24 near-isogenic lines, and they also showed different levels of virulence, where the anti-stain frequency of Pi-20 was the highest up to 54.35%. Moreover, the optimal combination of resistant genes was Pi-20×Pi-ks (RAC=0.28, PAC=0.23). [Conclusion] The virulence of M. oryzae was strong and 24 blast-resistant monogenes were nearly diseased in Taojiang rice blast nursery of Hunan Province. At present, rice cultivars carrying resistant genes combinations (Pi-20 with Pi-k, Pi-ks, Pi-3) can be popularized in Hunan Province, but new blast resistance genes must be further imported.

Abstract:[Objective] To explore synergy mechanism of cellulose biodegradation and relationships among the bacterial consortium, we took an approach of artificial constructing composite consortia. [Methods] Some strains were isolated from a microbial community which could ferment lignocellulose to produce biogas at high temperature. One of strains was identified as Bacillus licheniformis by sequencing nearly complete 16S rRNA gene. The strains’ combination, which consisted of the Bacillus licheniformis and Clostridium thermocellum strain CTL-6, had strong filter paper cellulose degradation ability. [Results] Throughout the 9-day co-cultivation, the cumulative degradation amount of filter paper was 484.6 mg and relative degradation ratio was as high as 93.2%. Overall, the variation of pH decreased firstly and then gradually increased. The initial pH of the culture solution was 7.00. The pH dropped to the lowest value (about 6.57) in 3 d. At the end of the culture period (9 days), the pH was 7.73. The combination could produce cellulase and hemicellulase, and two kinds of enzyme activity all represented the rising trend. The maximum of cellulase activity and hemicellulase activity were 0.32 and 0.57 U/mL, respectively, on the day 9. Lactic acid, formic acid, acetic acid, propionic acid and butyric acid were detected by HPLC during the co-culture. Among the five organic acids, the propionic acid and butyric acid had higher metabolism yield and the maximum concentrations were 1 068.8 and 1 477.3 mg/L, respectively. In addition to propionic acid, the concentration change trends of other 4 organic acids had no significant correlation with the change of the filter paper degradation. The total concentration variation of the five organic acids was in accordance with the variation of pH. This result indicated that it probably existed some not-detected acidic substances, the concentration variation of which played a decisive role in the pH variation of the co-culture system. [Conclusion] Bacillus licheniformis could effectively promote the cellulolytic activity of Clostridium thermocellum CTL-6, and the strains’ combination could also be used to artificially construct composite microbial which was able to convert cellulose to produce methane.

Abstract:[Objective] An antagonistic bacterium Y3F is isolated from marine algae in Lianyungang, its biocontrol effect on cucumber fusarium wilt is studied. [Methods] Y3F is identified as Bacillus cereus through the morphological, physiological and biochemical characteristics and phylogenetic analysis based on 16S rRNA gene sequence. The antimicrobial activity of broth and sterile filtrate of Y3F is checked by plate confrontation method, and its biocontrol activity to fusarium wilt of cucumber is checked by pot experiments. [Results] Y3F is identified as a member of Bacillus cereus. The sterile culture filtrate of Y3F in 2216E medium strongly inhibit the growth of Fusarium oxysporum strain cfcc 82185, implying that Y3F might secrete some kinds antifungal substances. The results of pot experiments indicated that soaking seeds and irrigating roots treatment (JG) could significantly increase the production of cucumber biomass, lower the incidence rate of fusarium wilt disease up to 50.46% after planting 30 days, and increase the total number of bacteria and actinomycetes, and decrease the number of fungi and Fusarium oxysporum strain cfcc 82185 in rhizosphere soil. [Conclusion] Strain Y3F can effectively control fusarium wilt of cucumber and improve microbial community structure in rhizosphere, and it has potential development and application prospect.

Abstract:[Objective] To confirm the pathogeny that causes Carassius auratus gibelio death in some fish farms in Shanghai, and characterize the virulence genes of the pathogenic bacteria. [Methods] The pathogenic bacteria were confirmed based on the pathogens screening and artificial challenge experiment, and identified according to 16S rRNA, gyrB and rpoB genes sequence and biochemical characteristics. Published nucleotide sequence methods were used to study the virulence genes (fimA, citC, gad, mukF, katB, esrB and sodB) by PCR amplification, and PCR primers for virulence genes. [Results] Strain GY15 isolated from infected ?sh was proved to be the pathogeny to Carassius auratus gibelio. Analysis of the 16S rRNA, gyrB and rpoB gene sequences of the GY15 gave the highest identity (99%) to Edwardsiella tarda. This was further supported by phylogenetic tree and biochemical characteristics. GY15 was identi?ed as E. tarda. Intraperitoneal injection with GY15 could lead to Carassius auratus gibelio death, with a value of the median lethal dose (LD50) of 4.26×105 CFU/ml. GY15 gave positive PCR results for 7 virulence genes. Antibiotic sensitivity test showed that among 35 antibiotics tested, GY15 was sensitive to 15 antibiotics such as enrofloxacin, florfenicol and ofloxacin, but was resistant to 18 antibiotics such as neomycin, acheomycin and cotrimoxazole. [Conclusion] Carassius auratus gibelio was the susceptible host species of E. tarda, which is a new potential threat to carp’s breeding.

Abstract:[Objective] We isolated, identified and tested antibiotic sensitivity of a pathogen from naturally infected Ictalurus punctatus. [Methods] The pathogenic bacteria were isolated and purified from lesions, liver, kidney and spleen of Ictalurus punctatus. Strain k1 was identifies using the biochemical identification and 16S rRNA gene sequence determination. The artificial infection test was done, and antimicrobial susceptibility was tested by disc diffusion method. [Results] Strain k1 was the pathogen of Ictalurus punctatus, and the LD50 of the isolates was 2.82×105 CFU/g. According to morphological and biochemical characteristics as well as the result of 16S rRNA gene sequence analysis, strain k1 was identified as Proteus vulgaris. Strain k1 was susceptible to 12 agents including ciprofloxacin, cefazolin, cefadroxil and other antibiotics. Meanwhile, it showed resistance to oxacillin, amoxicillin, furazolidone and other 7 antibiotics. [Conclusion] our results demonstrated that Proteus vulgaris was the pathogen causing high mortality in Ictalurus punctatus, and the disease may prevent by using drugs such as gentamicin and neomycin in fisheries.

Abstract:[Objective] As one of the key enzymes of the glycolysis in E. tarda, GAPDH was previously identified to be a broad-spectrum antigen and could be used as the target of bacterial vaccine design in aquaculture. We explore the mechanism of secretion of GAPDH in E. tarda. [Methods] Secretion of GAPDH in E. tarda EIB202 and mutants of several secretory pathways were analyzed by Western blot and ELISA. The levels of GAPDH in the supernatants of mutant library were analyzed through high-throughput screening. Quantitative real-time PCR was used to compare the mRNA expressions of mutants obtained from screening. [Results] Secretion of GAPDH in E. tarda is associated with classical secretion systems. Through high-throughput screening, two mutants ΔesrA and ΔesrC were found to have higher levels of GAPDH in the supernatants. Lacking of these two genes lead to significant up-regulation of secretion of GAPDH. [Conclusion] EsrA and EsrC both played a negative regulation role in GAPDH secretion.

Abstract:[Objective] In recent years, the epidemic of infectious bronchitis virus in China has become a popular trend in domestic flocks, especially the emergence of mutant strains, which aggravate the harm to the flocks. Major histocompatibility complex (MHC I) class I is able to bind antigens containing certain motif and further recognize and eradicate cells affected by virus. Identification of the binding motif of the complex of chicken MHC I protein BF2*15. [Methods] The complex of chicken MHC I protein BF2*15 and Nucleoprotein of infectious bronchitis virus (IBV) octapeptide were constructed by computational method, including homology modeling, molecular dynamics simulation and molecular docking. [Results] According to the interaction relationship of BF2*15 and IBV Nucleoprotein peptide, we conclude that “x-Arg-x-x-x-Arg” may represent a potential binding motif of BF2*15. [Conclusion] Interactions of the major histocompatibility complex class I BF2*15 and cytotoxic T lymphocyte epitopes from nucleoprotein of avian infectious bronchitis virus was illuminated in this study, and the results may shed light on the understanding of immune mechanism of IBV and research towards universal IBV vaccine.

Abstract:[Objective] Pseudomonas chlororaphis GP72 is a plant growth-promoting rhizobacterium. Its secondary metabolite 2-hydroxy-phenazine (2-OH-PHZ) has broad-spectrum antimicrobial activity, although its yield is too low to apply in agricultural pest management. [Methods] The limiting factor PhzO in 2-OH-PHZ biosynthesis pathway was replaced by green fluorescent protein (GFP), and a novel atmospheric and room temperature plasma (ARTP) technology was used to mutagenize the strain. High-throughput screening of the mutants in a 96-well plate was done by using a microplate reader. Finally, GFP was replaced reversibly by PhzO in the strain with high fluorescence intensity to obtain the 2-OH-PHZ high-yield mutant. [Results] The yield of 2-OH-PHZ was 4.62 times higher than that of the wild-type strain in KB medium in shake flask culture. [Conclusion] The high yield of 2-hydroxy-phenazine GP72 mutants could be obtained rapidly and safely by high-throughput screening based on ARTP mutagenesis using GFP substitution of the restriction factor as the marker. This method overcomes the shortcomings of traditional breeding methods. This method could be used as a reference for other microbial breeding.

Abstract:[Objective] To identify the inositol polyphosphate kinase Kcs1 in Candida albicans and study the role of Kcs1 in autophagy, hyphal growth and virulence. [Methods] The mutant kcs1Δ/Δ and the reconstituted strain KCS1c were constructed by homologous recombination. Transport and degradation capability of the autophagy-related protein Atg8 were determined by fluorescence microscopy and Western blotting under nitrogen starvation. Hyphal development ability was assayed using hypha-inducing medium. The virulence of C. albicans was determined using the macrophage model and the mouse systemic infection model. [Results] Nitrogen starvation experiment and GFP-Atg8 analysis revealed that deletion of KCS1 caused defect in transport of Atg8 to vacuole and degradation of Atg8 in the vacuole. Moreover, the kcs1Δ/Δ mutant attenuated the ability of hyphal development. In addition, the kcs1Δ/Δ mutant decreased the ability of fighting against microphage attacks. However, the ability of systemic infection was not impaired by deletion of KCS1. [Conclusion] The inositol polyphosphate kinase Kcs1 played an important role in various physiological processes in C. albicans, including autophagy, hyphal development and macrophage sensitivity.

Abstract:Microbial secondary metabolites have been an important source of drugs and their leading compounds because of their novel chemical structures and various biological activities. However, in recent years, there is a high frequency of re-discovery and a low frequency of new discovery of active compounds from microbial pure culture. As microbial co-culture is closer to the natural environment of microbial growth, it plays an important role in new compound mining. Here, we review the progress of new compound mining through co-culture from fungi vs fungi and fungi vs bacteria, and the current problems of this field for future research is also mentioned.

Abstract:Phyllosphere contains a wide distribution of nitrogen-fixing bacteria that fix nitrogen for bacterial and plant growth. The properties of nitrogen-fixing bacteria have been partially studied through traditional culture-dependent methods. However, knowledge on nitrogen-fixing bacteria diversity, community structure and function in phyllosphere remains limited. Recently, developments in molecular ecology techniques have led to the widespread use of culture-independent methods, enable researchers to understand phyllosphere microbial diversity and ecological function. Phyllosphere nitrogen-fixing bacteria exhibit abundant diversity that is mainly affected by environmental conditions (temperature, humidity and light), the host plant species and microorganism interactions. Unlike rhizosphere diazotrophs, phyllosphere nitrogen-fixing bacteria may not be affected by chemical fertilizers and could thus be potentially beneficial in agricultural applications. This review discusses the latest research on phyllosphere nitrogen-fixing bacteria diversity and ecological functions in agricultural, forest, and marine ecosystems, and on the factors influencing the nitrogen-fixing system, thus providing new insight into phyllosphere nitrogen-fixing bacteria.

Abstract:Coronaviruses are enveloped viruses carrying single stranded and positive RNA genome. The viruses are important human and animal pathogens, causing acute or chronic symptoms in respiratory, nerve, liver, gastrointestinal tract. From 2000, epidemics of SARS (Severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome) in human population and worldwide epidemic of PEDV (Porcine epidemic diarrhea virus) in pig population have made coronaviruses the focus of scientific research. Coronavirus S protein is the key protein involved in receptor recognition and membrane fusion. The S protein plays an important role in virus tissue or host tropism and virulence. This article reviewed the recent studies on structure and function of coronaviral S protein, and the interaction between the S proteins and their receptors, aiming to provide more information for research on the viral invasion mechanism, reverse genetics as well as the development of receptor blocking drugs.

Abstract:Acinetobacter baumannii is a nosocomial pathogen, which garners widespread attention for its infections and drug resistance. Tools to rapidly dissect the A. baumannii genome will facilitate the study of its pathogenic mechanism. Here, we summarized genetic manipulation tools for A. baumannii genome, including various methods for transformation of exogenous DNA into A. baumannii (electroporation, natural transformation, conjugation) and genome modification techniques (allelic exchange, recombineering, transposon mutagenesis), and carried out a preliminary prospect on progress of genome editing tools for A. baumannii.

Abstract:In order to improve the effectiveness of practical teaching in Biotechnological Specialty, exploration and practice of a reform on exercitation mode was boldly performed to solve the three major problems existing currently, including few outside-school internship, specific-job training and scattered practice while too many inside-school exercitations, visits and centralized practice. A triune exercitation mode comprising “production practice+graduation practice+obtain employment” was established. A classified practice method containing three types of exercitations, which is designated “innovative, administrative, entrepreneurial” type, was implemented to join the employment of graduates. Moreover, an exercitation system achieving the tripartite win-win situation of school, internship units and students has been formed, and showed satisfactory effects in practice. These research results can provide reference for the practical teaching of undergraduates in universities.

Abstract:Environmental Microbiology, one of the basic professional courses, is helpful to figure out environmental issues and to remove engineering roadblocks. Environmental Microbiology was endowed with the support of the national excellent courses and the professional core courses in Zhejiang University. In the course construction process, a new content system was established targeting pursuing the answers of “what are microorganisms”, “what can microorganisms do” and “how to use microorganisms”. A new teaching pattern was proposed, including students lecture, special subject lecture, topic discussion, thesis course and comprehensive assessment. The practice of the new content system and teaching pattern resulted in a good reward, demonstrating the feasibility for the course.

Abstract:[Objective] To explore the molecular pathogenic mechanism of C. chrysosperma, a protoplast preparation and transformation method is established, and the transformation efficiency and the stability of transformants are analyzed. [Methods] In this study, strain CFCC 89981 served as the recipient. The protoplasts were prepared using cell wall degrading enzymes, and transformed by gGFP plasmid mediated by PEG. PCR amplification, Southern blot and fluorescent observation were used to confirm the transformation efficiency and the genetic stability of GFP-tagged transformants. [Results] High-quality protoplasts with excellent regeneration efficiency (63.74%±9.73%) were generated using Driselase and Lysing enzyme digesting fresh mycelium with 1.2 mol/L KCl in pH 5.5 for 4 h and transformed with the gGFP plasmid using PEG. A total of 304 hygromycin B resistant transformants was obtained though added 4 μg DNA. FDA staining results showed that 98% of protoplasts exhibited high activity. The GFP fragments were detectable in the genomes of transformants by both PCR amplification and Southern blot analysis, and the fluorescence detection results also indicated that the GFP gene had been integrated and was stably expressed in the C. chrysosperma genome. Highly intense green fluorescence was observed in single-spore purified transformants. The GFP gene and hph gene were stably expressed after subculturing in PDA plates without hygromycin B resistant. [Conclusion] The high quality and viable protoplast of C. chrysosperma preparation and transformation system are established, it will provide a solid foundation and greatly facilitate the future studies on functional genomics and pathogenic molecular mechanism in C. chrysosperma.

Abstract:[Objective] To prepare colon-targeted nanocarriers. [Methods] SOE-PCR methods were used to prepare TK-vp2(?vp2) by inserting TK peptide gene into porcine parvovirus VP2 structural protein of loop2 and loop4. Then, TK-VP2 protein was constructed, expressed and self-assembled in the Bac-to-Bac? baculovirus expression system. [Results] The ?vp2 gene obtained through SOE-PCR methods, Bacmid-?vp2 was constructed in the Bac-to-Bac? baculovirus expression system and the recombinant baculovirus was successfully constructed in Sf9 insect cells. The results of the direct immunofluorescence, SDS-PAGE and Western blot assays showed that the ?VP2 proteins were expressed in the Bac-to-Bac? baculovirus expression system and the protein was approximately 70 kD. The transmission electron microscopy (TEM) results indicated that the ?VP2 proteins can self-assemble to form virus-like particles (TK-VLPs) ranging between 20 and 30 nm. [Conclusion] TK-VLPs nanocarrier was obtained, providing a basis to further study the feasibility of TK-VLPs as colon targeting nanoparticle.