Abstract:[Objective] The aim of this study was to obtain a more stable cephalosporin C (CPC) acylase by screening site-directed saturation mutation libraries of Pseudomonas sp. SE83 acyII and to characterize the structure and function of mutant enzyme. [Methods] We computated B factor based on the structure of Pseudomonas diminuta N176, a homologue of Pseudomonas sp. SE83 acyII, and constructed site-directed saturation mutation libraries of SE83 acyII. Combined with pH indicator assay, we used a Biomek FXP automation workstation high-throughput screening method to screen more stable CPC acylase. The enzyme properties were further defined by comparison with the wild type enzyme. The relationship between structure and function of the positive mutants was studied by homology modeling, using SWISS-MODEL software. [Results] We found 9 targeting sites by B factor homologous structure alignment and computation. After 3 rounds of screening, we found that the mutations occurring at residues R218 and K226 could enhance the thermostability of acylase, and the most stable mutants were identified as R218Q and K226V. Their half-lives at 40 °C were approximately 3.77- and 2.77-fold of the wild type enzyme, respectively. The catalytic efficiency kcat/Km was also approximately 1.8- and 3.1-fold of the wild type, respectively. The possible mechanism for the enhanced stability might be the increments of hydrogen bonds and hydrophobic interaction, which were analyzed by homology modeling. [Conclusion] It proved to be an efficient and reliable strategy for improving enzymatic stability based on B factor, and the positive mutant R218Q and K226V could enhance the stability and catalytic efficiency of CPC acylase. Thus, this study may serve as a useful reference for further improving enzyme properties.

Abstract:[Objective] To discover novel esterase from soil microbes by metagenomic approach. [Methods] Tributyrin-containing LB agar plates were used to screen a metagenomic library and the predictive esterase gene on the positive clone was heterologously expressed for biochemical analysis. [Results] We successfully found a positive clone from a soil microbial metagenomic library that comprised of 120 000 clones by function-based screening. Then the putative esterase gene (estyn1) of this clone was heterologously expressed and its properties including optimum pH, optimal temperature, and tolerance to organic solvents and metal ions were studied. Its optimum pH was 9.0 and its optimal temperature was 56 °C. The esterase activity was enhanced in low concentration of organic solvents, especially in DMSO (dimethylsulfoxide, activity enhanced to 144% at 10% DMSO). [Conclusion] Novel enzymes could be discovered by metagenomic approach. ESTYN1, with good tolerance to organic solvents and high temperature, would be a promising enzyme for industrial production.

Abstract:[Objective] The mechanism of the generation of various kinds of poly-γ-glutamic acids (γ-PGA) from Bacillus licheniformis WBL-3 affected by Mn2+ was investigated in this study. [Methods] Three L- and D-glutamic metabolism related enzymes, glutamate racemase (GR; encoded by racE), D-alanine aminotransferase (D-DDT; encoded by dat) and glutamine synthetase (GS; encoded by gltA), were cloned and expressed meanwhile B. subtilis KH-2 was used as the control strain. The mechanism synthesis of γ-PGA by different concentration of Mn2+ was studied using the methods of enzyme activity, combined with transcriptional analysis. [Results] When the concentration of Mn2+ varied from 0 to 0.6 mmol/L, the proportion of D- isomer increased from 22% to 67%. The catalytic activity (kcat/Km) of GR in Bacillus licheniformis WBL-3 with 0.6 mmol/L Mn2+ is higher than that without Mn2+. The GS activity was measured only in the presence of Mn2+. RT-PCR study showed that the transcription ratio of racE, dat and gltA with 0.6 mmol/L Mn2+ to those without Mn2+ was 2.16, 4.44 and 1.84-fold, respectively. [Conclusion] Mn2+ activated the expression of GR, D-DDT and GS, promoted the metabolism of L-glutamic acid, increased the proportion of D-glutamic acid, and increased the proportion of γ-D-PGA.

Abstract:[Objective] We constructed engineered bacterium to improve diesel biodegradation and to study the effect of p450 gene on diesel biodegradation. [Methods] The p450 gene of Alcanivorax borkumensis SK2 was constructed in pCom8 and the p450-SK2/pCom8 plasmid was transformed into Escherichia coli DH5α. The expression of AlkB protein in E. coli DH5α was detected using SDS-polyacrylamide gel electrophoresis. The recombinant plasmids expressed rightly were transformed into diesel-degrading Acinetobacter sp. strain Y9. The degrading characteristics and the expression of p450 in engineered bacterium p450-SK2/Y9 were studied. [Results] The identifying results by PCR and digestion with Sal I and Nde I indicated that the recombinant plasmids and the engineered bacterium p450-SK2/Y9 were constructed rightly. When the induction concentration of diesel was above 1% (v/v), the expression level of p450 gene was higher and increased slightly with the increasing of induction time. Diesel degradation ratios of p450-SK2/Y9 were not higher than that of Y9 when it was inoculated solely, but the p450-SK2/Y9 could facilitate the biodegradation of diesel in bacterial consortium. The results of SDS-PAGE showed that p450 gene could be expressed p450-SK2/Y9, and the expression level was higher in the bacterial consortium than that of single bacterium. [Conclusion] The engineered bacterium p450-SK2/Y9 could facilitate the biodegradation of diesel in bacterial consortium, and the expression level was higher in the bacterial consortium than that of single bacterium. These results are useful to improve diesel biodegradation and study on the mechanism of p450 gene for diesel biodegradation.

Abstract:[Objective] The anti-microbial ability of residual allicin in garlic wastewater results in its low bio-degradability. To resolve this problem, this paper tried to isolate the allicin-degrading bacteria and to further analyze its degradation character. [Methods] We inspected the bacterial diversity in the polluted sediment of garlic wastewater by 16S ribosomal RNA gene sequencing, and 16 microbial species were found which suggested allicin degradation bacteria might be included. Next, our research was focused on enrichment, separation and characterization of the allicin-degrading strain. [Results] An allicin-degrading strain JX6-2 was isolated from the sediment of allicin wastewater, and identified as Weissella. The strain could use allicin as the only carbon source and grow on the range of allicin concentrations (50 to 300 mg/L). The optimal pH and temperature for allicin degradation were determined to be 7.0 and 35 °C, respectively. The presence of glucose as an additional carbon source (up to 1 000 mg/L) had no adverse effect on bacterial growth and allicin degradation. [Conclusion] We have isolated an allicin-degrading strain and optimized its degradation conditions. It has a potential to be applied in the practical treatment of aniline-containing wastewater.

Abstract:[Objective] We studied the stability properties of the biosurfactant produced by Pseudomonas aeruginosa. And we also investigated the enhancement role of this biosurfactant in the biodegradation of anthracene by Irpex lacteus F17. [Methods] The biosurfactant was extracted using trichloromethane from the fermented broth of Pseudomonas aeruginosa. In order to analyze the stability of the biosurfactant, we determined the surface tension values of the biosurfactant under different conditions using surface/interface tensiometer. During the biodegradation of anthracene by Irpex lacteus F17, we added the biosurfactant to investigate the enhancement role of this biosurfactant. [Results] The result showed that the Critical Micelle Concentration (CMC) of this biosurfactant was 40 mg/L. We observed good stabilities of this biosurfactant in the range of 15?150 °C and pH 6.0?13.0, and this biosurfactant could bear high salt concentration. Using biosurfactant could enhance the biodegradation of anthracene greatly. The degradation rate of 82.9% was obtained after 15 days using 50 mg/L biosurfactant. The biodegradation was enhanced more efficiently when the biosurfactant was added one day before inoculum of degrading strain Irpex lacteus F17. Compared with chemical surfactants, biosurfactant exhibited a more excellent efficacy in enhancing the biodegradation of anthracene. [Conclusion] The excellent surface properties and stabilities and more efficient enhancement in the biodegradation were benificial to the application of biosurfactant in bioremediation.

Abstract:[Objective] In this study, the distribution of Spirulina (Arthrospira) during July to September 2015 in Bohai wetland and its surrounding waters was researched. Bohai has a long coastline and relatively diverse types of wetland which provides perfect conditions for the study. Moreover, the correlation between the distribution of Spirulina (Arthrospira) and environment factors was analyzed. [Methods] The relationship between the number of Spirulina (Arthrospira) and environmental factors was analyzed through correlation and grey relational analysis. [Results] The number of Spirulina (Arthrospira) was dramatically and positively related to pH, salinity, TN, TP, COD and NH3-N (P<0.05, P<0.01) while was significantly and negatively correlated to DO (P<0.05, P<0.01). The number of Spirulina (Arthrospira) was negatively correlated with temperature as well, to which the correlation was not significant (P>0.05). The result of grey relational analysis demonstrated that the order of the correlation degree of various environmental factors on the number of Spirulina (Arthrospira) had following orders: COD>pH>TN>temperature>TP>DO>salinity>NH3-N. [Conclusion] Spirulina (Arthrospira) widely existed in Bohai coastal wetland and its surrounding waters. The environmental factors that had the most and the lest influence on the distribution of Spirulina (Arthrospira) were COD and NH3-N, respectively.

Abstract:[Objective] Protein phosphorylation plays an important role in many important cellular processes, such as the extracellular cellulase induction signal sensing and intracellular signal transduction processes in filamentous fungi. The protein phosphorylation is controlled by protein kinases. [Methods] to discover the roles of protein kinases in cellulase induction signaling pathway, we analyzed cellulase expression levels of 61 kinase mutants using 2% crystalline cellulose as the sole carbon source. Compared with the wild type, cellulase productions in 7 mutants were significantly changed. Then, we did the SDS-PAGE analysis and measured the endo-beta-1,4-glucanase activity, beta-glucosidase activity, cellobiohydrolase activity, xylansae activity of these 7 mutants. [Results] We found that extracellular proteins of mutants W14, W38, W87 and W40 increased more than 30%, and their endo-beta-1,4-glucanase activities increased by 62%, 42% and 42% respectively, except for mutant W14. Moreover, the extracellular proteins of mutants W85, W26 and W46 decreased over 50%, and their endo-beta-1,4-glucanase activities were also reduced by 86%, 75% and 84% respectively. [Conclusion] These observations about serine/threonine protein kinase genes in Neurospora crassa could be helpful to better understand the roles of protein kinases in cellulase induction pathway.

Abstract:[Objective] To study the molecular mechanisms underlying improved acetic acid stress tolerance of the flocculating yeast Saccharomyces cerevisiae SPSC01 by zinc sulfate supplementation. [Methods] Global gene expression profiling was studied by comparative transcriptomic analysis using yeast cells of the log phase cells that were grown with or without 0.03 g/L zinc sulfate addition in the presence of 10.0 g/L acetic acid. [Results] Transcription levels of 50 genes were up-regulated and 162 genes were down-regulated when zinc sulfate was added in the fermentation medium. Genes involved in carbohydrate metabolism, methionine synthesis, and vitamin biosynthesis were up-regulated. In addition, genes encoding the antioxidant enzymes and other stress responsive genes were also up-regulated. [Conclusion] Zinc sulfate addition affected global gene transcription of S. cerevisiae, exerting positive effects on the expression of antioxidant enzymes, stress tolerance and redox balance, as well as energy metabolism. Our results indicate that zinc sulfate supplementation improves acetic acid tolerance of S. cerevisiae by regulating multiple genes and metabolic pathways.

Abstract:[Objective] Autolysis is a physiological accommodation of bacteria to lyse themselves under stress conditions. The aim of this study is to investigate comprehensively the autolysis of the lactic acid bacteria (LAB) strains stressed by various growth inhibitors. [Methods] The methods include the autolysis assay of of LAB strains with various origins, as well as the determination of autolysis profiles of Lactococcus lactis MG1363 under the conditions specified by different growth media and inhibitors’ interference. [Results] The results showed that the autolysis of L. lactis MG1363 could be induced in glucose strictly-limited medium through the action of ampicillin (Amp), and this phenomenon could only occur at the glucose exhausting point, indicating a growth phase-dependent pattern. And concomitantly, the expression of four predominant autolysins was significantly altered to a variable extent upon the addition of Amp. Furthermore, all the tested inhibitors negatively affected MG1363’s autolysis under non-nutrient conditions, probably suggesting a mode of co-regulation between cell wall synthetic and hydrolytic enzymes. [Conclusion] Collectively, a significant discrepancy was proposed with respect to the autolysis of LAB strains administered with different inhibitors, and moreover, this peculiar phenomenon was strictly nutrition-and growth phase-dependent.

Abstract:[Objective] To elucidate the characteristics of a ferulic acid esterase catalytic domain within a XynZ protein from thermophilic Clostridium thermocellum to provide a basis for its application in bioenergy and other fermentation industries. [Methods] A prokaryotic expression vector for the ferulic acid esterase catalytic domain (FAE) within XynZ protein as well as for this catalytic domain adding an extra carbohydrate binding module 6 (FAE-CBM6) was constructed and expressed in E. coli BL21(DE3) cells, respectively. Then, the effects of temperature, pH, substrates, metal ions and the CBM6 module on the catalytic activity of ferulic acid esterase were estimated. [Results] The favorable pH for optimal catalytic activity of recombinant FAE or FAE-CBM6 protein ranged from 5.0 to 9.0, and the favorable temperatures being 50?70 °C. Furthermore, different metal ion had a positive or negative effect on catalytic activity of these two recombinant proteins. [Conclusion] Under the same reaction condition, FAE-CBM6 usually produced a higher catalytic activity than FAE, indicating that the CBM6 module plays a key role in improving the ferulic acid esterase activity.

Abstract:[Objective] We isolated and identified walnut blight pathogen from diseased walnut growing in Jiange county, Guangyuan city, Sichuan province, to control the disease. [Methods] A total of 30 infested branch samples at different developing stages were randomly collected. Pathogen isolation and purification were conducted from walnut twigs tissue by normal tissue isolation method. To fulfill Koch’s postulates, pathogenicity test was done with the isolates. In addition, the pathogen was identified based on morphological characteristics and molecular biotechnology, and biological characteristics were determined using colony growth methods. [Results] In total 180 isolates were obtained. The pathogenicity test showed that the pathogens of walnut twig blight were mainly caused by Phompsis capsici. On PDA plate, the mycelium of the pathogen was white. The spore-bearing structures could generate oval and linear conidiums. PDA medium was the most appropriate for mycelium growth. The mycelium could effectively use a variety of carbon and nitrogen sources. Among the 9 carbon and nitrogen sources tested, the appropriate carbon sources were sucrose and fructose and the optimum nitrogen sources were molybdenum acid and phenylalanine. The best mycelium growth was obtained at 25 °C and pH between 6.0 and 7.0. The mycelium grew fastest under dark conditions. [Conclusion] Phompsis capsici can easily invade from wounds to cause disease.

Abstract:[Objective] This study aimed to determine the virulence of Nr5772 isolate of Metarhizium rileyi against larvae and pupae of Spodoptera litura, observe the development of M. rileyi in vivo and the physiological effect on host and illustrate the pathogenic mechanism of M. rileyi to insect pest. [Methods] Dipping method was adopted to assay the LC50 and LT50 of M. rileyi conidia against 3?6 instar larvae and pupae of S. litura. After the hyphal bodies of M. rileyi was injected into the hemocoel, the hemolymph was sampled at different time intervals to record the morphism, meanwhile, the number of hyphal bodies and hemocytes was counted and the phenoloxidase (PO) activity of host was assayed. [Results] As the lethal effect decreased along with the increase of instar, M. rileyi has the strongest lethal effect on 3rd instar larvae (LC50=3.12×106 spores/mL). The lethal velocity decreased with the increase of the instar and decline of spore concentration. When dipped in 5×109 spores/mL solutions, the 3rd instar larvae died rapidly (LT50=4.55 d). Within the following 64 h after inoculation, the hyphal body reproduction in vivo fit the power function model, whereas the hemocyte number in larvae was remain stable. At the primary period of infection (44 h post inoculation), PO activity in inoculated larvae was the same as control, however, it was inhibited strongly in the later stage (55?64 h post inoculation) when yeast-like hyphal bodies transformed into mycelia and killed the host. Apart from larvae, M. rileyi had no significant lethal effect on pupae. [Conclusion] Nr5772 isolate of M. rileyi has stronger lethal effect on earlier instar S. litura larvae, thus, to achieve better control efficiency, it is recommended to apply this fungus on lower instar larvae. M. rileyi has no obvious harmful effect on hemocytes and PO activity of host at the primary stage of the invasion, but it will strongly inhibit the PO activity at later stage.

Abstract:[Objective] To improve antimicrobial activity of Bacillus amyloliquefaciens strain PC2, the medium composition and fermentation conditions were optimized in our work. [Methods] Single factor experiments were carried to evaluate the effect of alternative constitutes for PDB medium on inhibition zone diameter against Staphylococcus aureus, Box-Behnken response surface methodology was conducted to obtain the optimal composition of the crucial medium factors. The optimal fermentation conditions were obtained through frequency analysis based on quadratic general rotary composite design. [Results] The optimal medium compositions were (g/L): potato 188.0, sucrose 22.0, L-monosodium glutamate 1.80, and the media cost was 0.81 RMB per litre. The optimal fermentation conditions were as follows: inoculation amount 6%, culture temperature 30 °C, liquid volume 40 mL/250 mL, rotation speed 185 r/min, initial pH value 7.0, and fermentation for 24 h. Appling optimal medium and culture conditions, the inhibition zone diameter of free-cell fermentation broth on Staphylococcus aureus reached to 30.82 mm, increased by 12.60 mm compared with the original conditions. [Conclusion] The present study provides a cost-effective medium and fermentation conditions for further study on large scale fermentation for antimicrobial substances production.

Abstract:[Objective] A new entomogenous fungi (GZUIFR-lgs-1), which was parasitic on grub larvae was identified and described. [Methods] The analysis mainly based on phenotypic characteristics and molecular phylogeny. [Results] The strain distinguished from the other Hirsutella species by phialides solitary, hyaline, occasionally proliferation and near right angle to hyphae, with distinctly swollen and verrucose base (19?27) μm×(2.7?3.6) μm, twisting in helices at the apex, neck (12.0?14.5) μm long; Conidia hyaline and smooth in mucous sheath, like orange segments (4.8?6.0) μm×(2.4?3.6) μm, single or occasionally in pairs. The result of phylogenic analysis based on ITS loci supported the morphological identification. [Conclusion] The specimen GZUIFR-lgs-1 is a new species in the genus Hirsutella, supported by the phenotypic traits and molecular analysis, named Hirsutella leigongshanensis.

Abstract:[Objective] The aim of this study was to explore the changes of soil bacteria and fungal diversity with strawberry rhizosphere soil of different continuous cropping years, to provide theoretical basis for the control of strawberry continuous cropping obstacles. [Methods] The soils without continuous cropping (CK), continuous cropping for 1 year (1Y) and continuous cropping for 8 years (8Y) were separately mixed with perlite in a ratio of 3:1. ‘Ningyu’ strawberry plants were transplanted in the potted soil on 10th of September in 2016; soil samples were collected 50 d after transplanting (before flowering); genomic DNA was extracted, and PCR was amplified to establish libraries. The 16S rRNA genes V4+V5 regions of soil bacteria and fungal ITS1+ITS2 regions were sequenced by Illumina high-throughput sequencing technology on Miseq platform, and related biological analysis was conducted to explore the changes of soil bacterial and fungal abundances, diversities and structures. [Results] A total of 3 192 bacterial operational taxonomic units (OTUs) and 762 fungal OTUs were obtained from 9 strawberry rhizosphere soil samples. Among them, Proteobacteria, Fimicutes, Actinobacteria, Bacteroidetes and Cyanobacteria were the dominant bacteria, which abundances of total bacteria were 87.86%, 64.83% and 61.79% respectively in strawberry rhizosphere soil of CK, 1Y, 8Y. Ascomycota and Basidiomycota were dominant fungi, which abundances of fungal community were 69.17%, 69.06% and 76.18% respectively in strawberry rhizosphere soil of CK, 1Y, 8Y. At phylum level, the ratios of Acidobacteria, Actinobacteria, Bacteroidetes, Fimicutes, Chloroflexi, Cyanobacteria, Planctomyces, Gemmatimonadetes, Proteobacteria, Ascomycota, Zygomycota and Chytridiomycota were significantly changed in different cropping years (p<0.05), The analysis at the genera level also showed that the ratios of 29 genera of bacteria and 19 genera of fungi were significantly changed in different cropping years (p<0.05). [Conclusion] With the extension of continuous cropping years,?ratios of various kinds of bacterial and fungi in ecosystem of strawberry rhizosphere soil will be changed significantly.

Abstract:[Objective] To isolate and characterize the lytic phage from fecal samples of dairy cattle in Shihezi, Xinjiang. [Methods] The phage was isolated and purified from dairy cattle fecal samples by double-layer agar plate method. The purified phage was concentrated negatively stained with uranyl acetate and observed by transmission electron microscopy. Meanwhile, the genome of the isolated phage was sequenced. Its genetic and evolutional history were analyzed. In addition, the host range, optimal multiplicity of infection (MOI), one-step growth curve, temperature and pH stability of the phage were investigated. [Results] One strain of phage that lysed Escherichia coli was isolated and named vB_EcoM_XJ2. The phage produced circular, not clear plaques with 0.7 mm–1.2 mm diameter. The electron microscope observation showed that the phage had a symmetrical Polyhedra the head and a contractile tails. The genome of the phage was comprised of double strand DNA, and the size was 75.617 kb with G+C% content of 42.09%. The homology of amino acid sequence of vB_EcoM_XJ2 was 94% with Escherichia coli phage NJ01 and vB_EcoP_SU10. The isolated phage was lytic to many strains of Escherichia coli, isolated from bovine mastitis milk samples. The phage could withstand the temperature up to 60 °C and keep stable titer under pH 5.0–11.0. The optimal MOI was 0.1, the incubation period was 15 min, the burst phase time was 95 min, and the burst size was 10.6 PFU/cell. [Conclusion] The newly isolated phage, named vB_EcoM_XJ2, belongs to lytic phage of Myoviridae, which shows strong capacity to adapt different environments conditions such as temperatures and acid-base values.

Abstract:[Objective] To explore the immunological responses of common carp (Cyprinus carpio) after being vaccinated with Aeromonas veronii ghosts and formalin inactivated A. veronii vaccine. [Methods] The experiment was consisted of three treatments and conducted in cylindrical fiberglass tanks (260 L/tank): CLGs group injected with CL0901 ghost vaccine, FKC group with formalin killed cell vaccine and the control group with phosphate buffer solution (PBS). All treatments were injected on 1st, 15 th and 29 th day after the experiment started. Three fish in each tank were sampled for blood and tissue at every 7 days after the first inoculation. The evaluation parameters included the serum antibody titer, relative percent survival (RPS), lysozyme (LZM), respiratory burst, complement protein 3 (C3), myeloperoxidase (MPO), malonic dialdehyde (MDA) and relative expression content of hepcidin gene. [Results] After the secondary inoculation, the antibody titer in CLGs group reached 26?28, and it was significantly higher than that in other groups (P<0.05). The respiratory burst, serum LZM, MPO and expression of hepcidin gene of CLGs group were significantly higher than those of PBS group (P<0.05). C3 in CLGs group was significantly lower than that in PBS group on 35 and 42 d (P<0.05). The MPO and expression of hepcidin gene of FKC group were significantly lower than those of CLGs group (P<0.05). The RPS was 57.70% in CLGs group, but 30.77% in FKC group. [Conclusion] These results indicated that the CLGs could enhance the serum antibody titer, respiratory burst, lysozyme, myeloperoxidase and relative expression content of hepcidin gene. As a whole, the effect of immune protection of CLGs is better than that of FKC.

Abstract:[Objective] To study the effect of inactivating MetD transporter system of Escherichia coli W3110 on the production of L-methionine. [Methods] We compared the metNIQ genes’ expression ratio of wild type E. coli W3110 and the deregulation of MetJ repression strains by Quantitative real-time PCR (RT-qPCR). The methionine uptake capacity of E. coli W3110 and Me05 were analyzed. To construct the inactivation of MetD transporter system mutants, metNIQ genes cluster, metN, metI, and metQ were deleted by Red recombinase system respectively. We analyzed their effects on the uptake capacity and methionine production of these mutants. [Results] The metNIQ genes expression ratio and the uptake capacity of methionine were significantly increased by the deregulation of MetJ repression. By deleting the metNIQ genes cluster of E. coli W3110 and Me05, the MetD transport capacity were inactivated which resulted in the reduced uptake capacity of methionine. Besides, we deleted the metNIQ genes cluster, metN, metI, and metQ of methionine-producing chassis strain Me06. Growth curves and flask batch fermentation showed that, the growth and production of methionine were improved by the deletion of metI. The methionine production improved from 0.39 g/L to 0.45 g/L, increased by 15.4%. The methionine yield on biomass improved from 0.14 to 0.15 g/g DCW. [Conclusion] Inactivation of the function of MetD transport system in E. coli could lead to the decreased uptake capacity of methionine. Deletion of the metI can improve the methionine-producing capacity of recombination E. coli strain.

Abstract:[Objective] Xianggu (Lentinulaedodes) is the second biggest edible fungi in the world. Genetic diversity and population structure analysis and reliable identification of strains are prerequisites for new cultivar breeding and the sustainable development of Xianggu mushroom industry. [Methods] Using polymorphic SSR markers to analysis the genetic diversity and population structure of the major cultivars in China, comparing their genetic pedigree and constructing the fingerprint profiles that can be used for varietal identification. [Results] In this study, 51 strains were identified with SSR markers including 24 pairs of primers and their polymorphism was 100%. According to the chustering analysis, experimental population could be divided into four clusters at the similarity of 0.69. Wild strains or cultivars derived from wild strains in cluster III and IV, and other hybrid cultivars in cluster I and II. The cultivar population can be divided into 6 genetics compositions according to the population structure analysis. Showing that several core strains are involved in others’ breeding procedure such as L808、L135, and can explain the pedigree of the cultivars used them as parents. 9SSR primer pairs delineated 45 of the cultivars based on the unique multilocus SSR fingerprint profiles. [Conclusion] The results indicated a close genetic relationship of cultivars in China, and the breeding research was always focus on several core strains such as L808、L135、9015. This research offered some references to the genetic breeding that has our own intellectual property and can adjust different cultivation mode. The fingerprint profiles could also provide safeguards for the reliable identification of Xianggu strains.

Abstract:[Objective] This study aimed to synthesize novel thiourea derivatives with good antimicrobial activities. [Methods] Thiourea derivatives were synthesized through condensation of various primary amines with the α-isothiocyanatoacrylic ester (ICE) intermediate; their structures were determined by MS and NMR analysis and their antimicrobial activities were then evaluated. [Results] Six novel thiourea derivatives and one new α-isothiocyanatoacrylic ester derivative were synthesized through condensation of corresponding primary amines with α-isothiocyanatoacrylic ester. The bioactivities of these compounds were tested against several representative pathogenic bacterium and fungus strains. Specifically, the thiourea derivatives showed considerable inhibition activities against Cryptococcus neoformans, the pathogen fungus of cryptococcosis. [Conclusion] Synthesis of new thiourea derivatives and test their biological activities is a potential way to discover small molecule drug leads.

Abstract:[Objective] To isolate and characterize ocular Chlamydia trachomatis from trachoma samples of Tibetan children from Qinghai province. [Methods] Swabs of left and right conjunctivas and conjunctival sacs were collected into one milliliter of transportation media. Fifty microliters of each sample were used to infect BGM cells by using centrifugation. Infected cells were cultured at 37 °C for 72 h, and were passaged for three successive times. Chlamydial inclusions were observed using phase microscopy. Chlamydial cultures and clinical samples were characterized by using ompA gene sequence analyses. [Results] A total of 115 samples from 45 trachoma patients were collected. 54 samples were ompA-PCR positive and 15 samples were culture positive. All ompA genes of these samples belong to genotype B, are grouped into 3 variants and have a UGT-type codon. Chlamydial isolates QH111L and QH111R were from left and right conjunctival samples of No. 111 patient, respectively, but their ompA genes have a nonsynonymous base difference. This single nucleotide change was only found in left conjunctiva of No. 111 patient, suggesting that QH111L may be a newly emerged ompA variant. [Conclusion] 15 ocular Chlamydia trachomatis strains were isolated from Tibetan children of Qinghai. All strains belong to genotype B and three different ompA variants. Isolates from a same patient’s separate conjunctivas were found to have different ompA genes. Identification and characterization of these newly isolated strains will further our understanding of trachoma epidemiology and evolution.

Abstract:Citrinin is a nephrotoxicity fungal metabolite produced by several fungi of the genera Penicillium, Aspergillus and Monascus, and contaminates food and feed. Citrinin synergistically combines with other mycotoxins, such as patulin and ochratoxin to cause great harm to human and animal health. This article reviews the scope of citrinin toxicity and the progress of different methods to control citrinin in food and feed.

Abstract:Akkermansia muciniphila was isolated from human faeces in anaerobic medium containing gastric mucin as the sole carbon and nitrogen source. It represents approximately 1% to 3% of the total microbiota in the intestine of healthy adult, and it produces propionic acid as main metabolite. This mucin-degrading bacterium has been closely correlated with host health. A. muciniphila administration could enhance glucose tolerance, reduce insulin resistance, modulate pathways involved in establishing homeostasis for basal metabolism and immune tolerance toward commensal microbiota. But on the contrary, the abundance of the mucin-degrading A. muciniphila was significantly increased by heme diet, which is associated with epithelial hyperproliferation, and destroyed mucus layer. The mechanisms of mucin utilization by this bacterium in the gut and the interactive mechanisms between A. muciniphila and host is still unknown and need further explorations.

Abstract:Gene recombination technology has become a main route to produce proteins in laboratory and industry. Though many proteins have been efficiently expressed in Escherichia coli, people have difficulties when target genes express at a very low level or produce inclusion body, especially for the proteins important for research and production. The expression level of molecular chaperones can be enhanced by the transcription factors of a heat shock or cold shock system in vectors pHsh and pEXC, which could reduce cytotoxicity of target proteins and formation of inclusion body. Thermostable enzymes used in biosynthesis, molecular modification or biodegradation could be overexpressed in pHsh expression system, while the genes from animals, plants and mesophilic microorganisms can be effectively expressed by using vector pEXC. The development of these novel vectors supply new strategies and effective approaches for small-scale preparation and industrial production of enzymes and bioactive proteins.

Abstract:This paper concludes totally more than 600 species in 170 genera of sulfur-circulating prokaryotes, including more than 150 species in 48 genera with thium, more than 20 species in 6 genera belong to green sulfur bacteria, nearly 100 species in 33 genera belong to purple sulfur bacteria, 56 species in 23 genera with sulfur, more than 210 species in 50 genera with desulfurization (sulfate reduction), and 3 species and 1 genus with sulfur dismutation. According to the properties of these sulfur-circulating prokaryotes, they are classified as sulfur oxidation, sulfur reduction and sulfur dismutation and of vital importance to sulfur circulation in nature.

Abstract:Changes in precipitation pattern and increase in regional nitrogen deposition have turned to be two hot issues in the study on global change in recent years. As grassland ecosystems are widely distributed over the globe and mostly in the ecological fragile zones, they are very sensitive and susceptible to the interference of global change and then lose balance. Soil microorganisms and soil enzyme activity are sensitive indicators of soil quality in natural ecosystems, They can be used to monitor changes of grassland ecosystems in structure and function under the grand background of changes in water and nitrogen. Based on this, a review is presented of progresses of the study on impacts of changes in precipitation pattern and increase in nitrogen deposition, and their interaction on number, biomass and diversity of soil microbes and enzyme activity, so as to provide a scientific basis for better prediction, evaluation and eventual control and maintenance of the stability of grassland ecosystems. At the same time, problems and shortcomings existing in the current work are analyzed and key scientific issues in future research are also discussed and previewed.

Abstract:Biofilm is a highly structured bacterial community state. Bacterial extracellular matrix substances such as EPS, eDNA, and extracellular proteins are involved in the formation of biofilm. These matrix substances enhance biofilm mechanical stability, promote bacterial adhesion to solid surface, facilitate nutrient circulation and gene transfer, and provide other advantages to the bacterial survival. Biofilm formation is related to quorum sensing, C-di-GMP and sRNA. In soil environment habitat plenty of bacteria, many of which colonize plant roots and thus interact with host plants intensively. Biofilm is known to play an important role in plant colonization by soil bacteria including both phytopathogenic and plant beneficial ones. In this paper the biofilm studies are reviewed with a focus on biofilm composition, regulation and, especially its association with plant roots.

Abstract:There are many shortcomings in the experiment course of Environmental Microbiology, such as the less practice on experiment skills, no relationship between the designed contents and scientific researches, and sometimes the valued experiments were ignored. Therefore, changes should be done urgently. In this paper, the current status and problems in the experiment course of Environmental Microbiology were totally analyzed. The comprehensive experiments were designed aiming for scientific research innovation and scientific research goals. The inovation idea was that experimental teaching results should be converted into scientific research pre-production. Moreover, in order to improve the experimental skills and creativities, the specific directions and methods of the teaching innovation was mainly discussed.