• Volume 47,Issue 1,2020 Table of Contents
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    • >Industrial Microbiology
    • Distribution of Lactobacillus sp. in Chinese liquor fermentation system from different producing location by three-step fluorescent quantitative PCR

      2020, 47(1):1-12. DOI: 10.13344/j.microbiol.china.190150

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      Abstract:[Background] Lactobacillus sp. is an important function microorganism in Chinese liquor fermentation. However, there is no quantitative method to uncover the distribution of Lactobacillus sp. among different production locations. [Objective] This study aimed to build a quantitative method to uncover the distribution Lactobacillus sp. among different Chinese liquor production locations. [Methods] Specific primers were designed based on 16S rRNA gene sequence, and the specificity was verified by PCR. The qPCR reaction procedure was further optimized to increase amplification efficiency and reveal the distribution of Lactobacillus sp. [Results] a pair of specific primers were designed and validated, its amplification product size was 445 bp. By optimizing annealing and extension conditions, a three-step qPCR quantitative method was developed with independent denaturation, annealing and extension process. The amplification linearity of this method was strong (R2>0.99). The sensitivity was high, the quantitative limit was 17.9 copies/μL. The repeatability was well, the coefficient of variation of Ct value was less than 1%. Following and detecting the representative fermentation system from 10 producing locations in China, Lactobacillus sp. presented in 8 producing areas. In the different making process samples from the same region, Lactobacillus sp. was detected but its content was significantly different. The content of Lactobacillus sp. from sesame aroma fermentation system in Weifang, Shandong province was the highest (7.27±0.04 lgcopies/g). The growth curve of Lactobacillus sp. in this system was divided into two stages: the growth stage (0 to 15 d) and the stable stage (15 to 45 d). [Conclusion] The established novel qPCR method can accurately quantify and identify Lactobacillus sp. Through follow-up detection, it was found that Lactobacillus sp. was widespread in Chinese liquor fermentation system, the distribution of Lactobacillus sp. was determined by the origin, the content of Lactobacillus sp. was affected by the making process. During fermentation process, the content of Lactobacillus sp. has an obvious dynamic characteristic. This study will improve the development of function study about Lactobacillus sp.

    • Engineering the yeast Yarrowia lipolytica to synthesize very- long-chain fatty acids and the effects of temperature on fatty acids synthesis

      2020, 47(1):13-23. DOI: 10.13344/j.microbiol.china.190090

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      Abstract:[Background] The yeast Yarrowia lipolytica is an oleaginous microorganism and can produce long-chain fatty acids and oils with high cell contents. However, the synthesis of very-long-chain fatty acids by this yeast remains to be investigated. [Objective] To engineer Y. lipolytica to synthesize very-long-chain fatty acids with high values and evaluate the influences of temperature on biomass, oil production, and fatty acids compositions. [Methods] The fatty acids elongation enzyme genes AtFAE1 in Arabidopsis thaliana, BtFAE1 in Brassica tournefortii and CgKCS in Cardamine graeca were codon-optimized according to the frequency of codon usage in Y. lipolytica. The plasmids pYLEX1-AtFAE1, pYLEX1-BtFAE1, pYLEX1-CgKCS and pYLEX1-AtFAE1-BtFAE1-CgKCS were constructed and used to transform the host strain Y. lipolytica Po1g, generating the yeast strains Po1g-AtFAE1, Po1g-BtFAE1, Po1g-CgKCS and Po1g-AtFAE1-BtFAE1-CgKCS. Furthermore, the DGAT1 gene encoding diacylglycerol acyltransferase was overexpressed in the strain Po1g-CgKCS to improve oil production. [Results] The results showed that the overexpression of AtFAE1, BtFAE1, and CgKCS led to the remarkably difference in the composition and content of fatty acid in Y. lipolytica. AtFAE1 mainly catalyzes the synthesis of C20:1, BtFAE1 prefers to synthesize erucic acid (C22:1), and CgKCS can catalyze the synthesis of nervonic acid (C24:1). Nevertheless, co-expression of the three genes in Y. lipolytica did not increase the production of nervonic acid. Overexpression of the gene DGAT1 in Po1g-CgKCS apparently increased the oil content by 50%. The results from the temperature experiment showed that a lower temperature benefited the synthesis of unsaturated fatty acids, while a higher temperature benefited the synthesis of saturated fatty acids in Y. lipolytica in our tests. [Conclusion] The fatty acids elongase CgKCS can directly catalyze the synthesis of the C24:1 fatty acid from the C18:1 fatty acid, and the synthesis of unsaturated fatty acids can be improved by optimizing the culture temperature. This study provides theoretical and technical guidance for the construction of cell factories for very-long-chain fatty acids and the optimization of fermentation.

    • Iron and manganese oxides enhance electron output efficiency of Clostridium pasteurianu

      2020, 47(1):24-34. DOI: 10.13344/j.microbiol.china.190191

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      Abstract:[Background] Fermentative dissimilatory metal-reducing bacteria, which are capable of reducing metallic oxides, get energy from fermentation. Little is known about how metallic oxides affect electron output efficiency of fermentative dissimilatory metal-reducing microorganisms. [Objective] This study was conducted to explore the influence of iron and manganese oxides (Fe2O3/MnO2) on electron output efficiency. [Methods] Different concentrations of Fe2O3/MnO2 were added into fermented system containing glucose and inoculated 5% C. pasteurianum. Electrochemical activity of C. pasteurianum was detected. The concentrations of Fe(Ⅱ) and Mn(Ⅱ) were measured by ferrozine spectrophotometry and formaldoxime method. Fermentation substrate and metabolites of C. pasteurianum were detected by gas chromatography and high performance liquid chromatography. Lastly, we calculated the electron output efficiency. [Results] The current density peaked with the value of about 0.93 mA/m2. The concentrations of Fe(Ⅱ) and Mn(Ⅱ) gradually accumulated. The consumption of glucose was increased by 9.4%/7.7%, Meanwhile, acetate production was increased by 37.5%/25.0%, and butyrate production was increased by 22.7%/6.8%. Additionally, hydrogen production was increased by 21.6%/9.8%, and the total electron output efficiency was increased by 24.27%/10.82%, respectively. The pH values between experimental group and control are no significant difference. [Conclusion] This study shows that iron and manganese oxides can improve the electron output efficiency of C. pasteurianum by increasing glucose consumption and buffering pH value. The results provide evidence for revealing the effects of multivalent metal oxides on the electron output of fermentative dissimilatory metal-reducing bacteria, and further expand our understanding of the interaction mechanism between multivalent metal oxides and fermentative dissimilatory metal-reducing bacteria.

    • >Marine Microbiology
    • Effective harvesting of the microalgae Desmodesmus sp. F51 with a novel bioflocculant

      2020, 47(1):35-42. DOI: 10.13344/j.microbiol.china.190462

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      Abstract:[Background] Cobetia marina can produce many extracellular products with flocculation activity and therefore is regarded as a novel bioflocculant. Desmodesmus sp. F51 is one of the best new resources for natural xanthophyll due to its ability to accumulate large amounts of lutein. However, up to date there is no report on the use of bioflocculant from Coberia marina for Desmodesmus sp. F51 harvesting. [Objective] The flocculation efficiency and mechanism of the new biological flocculant on Desmodesmus sp. F51 were investigated. [Methods] Effects of bioflocculant addition in different growth periods, the addition amount of bioflocculant, flocculation time and pH on flocculation efficiency were investigated. The functional groups of bioflocculant were analyzed. Zeta potential of Desmodesmus sp. F51 before and after the addition of bioflocculant under different pH conditions were measured, and the morphology of Desmodesmus sp. F51 under the microscope before and after the addition of bioflocculant were also analyzed. [Results] The highest flocculating efficiency (82.1%, 15 min, pH 8.0) was achieved when 2 mL of bioflocculant was added at the stationary growth phase of Desmodesmus sp. F51. Fourier transform infrared spectroscopy analysis showed the characteristic structure of polysaccharides and amide. Thus, it was speculated that the bioflocculant was mainly a mixture of heteropolysaccharides, containing a small amount of proteins. According to Bradford method, the protein content in the bioflocculant was about 0.4% (w/w) and the total sugar content determined by phenol-sulfuric acid method was about 34.5% (w/w), which was basically consistent with the results of fourier transform infrared spectroscopy analysis. The flocculating efficiency is above 60% when the pH value ranges from 4.0 to 11.0, which indicates that the flocculation efficiency is higher under both acidic and alkaline conditions. The analysis of Zeta potential indicates that the dominant flocculation mechanism of the bioflocculant may be adsorption bridging. [Conclusion] This study is of great significance for microalgae harvesting by bioflocculation.

    • >Environmental Microbiology
    • Degradation characteristics of a novel aniline blue-discoloring bacterial strain MP-13

      2020, 47(1):43-53. DOI: 10.13344/j.microbiol.china.190414

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      Abstract:[Background] The wide use of aniline blue, a common refractory organic pollutants, harms and threatens the ecological environments. Hence, it is important to develop an eco-friendly and cost-effective technology to treat the dye contaminated wastewater. Biological method is environmentally friendly and applied to deal with dye contaminated wastewater. [Objective] Our study was to provide a core theory and supporting technology to isolate and characterize a novel bacterial strain, Trabulsiella odontotermitis MP13, for its potential decolorization of dye wastewater. [Methods] Some aniline blue-decoloring bacteria were first isolated from the gut of a termite species, Microtermes pakistanicus, and then identified by 16S rRNA gene sequence analysis for their phylogeny property and other relevant biological properties. Further, with a variety of evaluation methods, such as FTIR and GC/MS, the degradation characteristics of aniline blue by strain MP13 were analyzed. [Results] A novel bacterial strain MP-13 was identified as Trabulsiella odontotermitis that could tolerate and effectively degrade aniline blue up to a high concentration of 1 500 mg/L. The optimal temperature, pH and rotation speed for dye decolorization were 30 °C, 8.0, and 180 r/min, respectively. The decolorization efficiency was up to 97.3%, when aniline blue concentration was at 200 mg/L. Further, the FTIR and GC/MS analysis showed that aniline blue was biotransformed into low molecular weight aromatic compounds. [Conclusion] Trabulsiella odontotermitis MP-13 exhibited a remarkable decolorization ability for aniline blue, suggesting its potential for industrial dye waste water treatment.

    • Transcriptomic analysis of Pseudomonas aeruginosa DN1 upon degradation of fluoranthene

      2020, 47(1):54-65. DOI: 10.13344/j.microbiol.china.190463

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      Abstract:[Background] Pseudomonas aeruginosa DN1, isolated from a petroleum-contaminated soil, exhibits a versatile metabolism and can utilize both n-alkanes and aromatic hydrocarbons. [Objective] The aim of this study was to fully understand the expression of vital genes for fluoranthene degradation and the changes of transcriptional spectrum of the DN1 in response to fluoranthene. [Methods] An RNA-Seq-based transcriptome was conducted for a global analysis of the DN1 in media with fluoranthene and glucose respectively, and all transcripts were subjected to KEGG classification and pathway annotation, GO classification and enrichment analysis. [Results] A total of 6 189 assembled contigs were obtained and analyzed in detail. In contrast to control group with glucose, 1 919 genes that corresponded to this degradation in the DN1 were found to be up-regulated and 1 603 genes were found to be down-regulated. KEGG pathway analysis showed that 112 KEGG pathways with up-regulated expression were variant, and 317 of the 1 408 genes that were about 73.4% of total differential genes annotated to the “metabolism” pathway involved in carbohydrate metabolism and xenobiotics biodegradation and metabolism, accounting for 16.53% of the “metabolism” pathway, which suggested that fluoranthene degradation by the DN1 may be closely related to these pathways. Moreover, the differentially expressed genes of primary metabolic pathways were identified and the results indicated that the main pathways, such as ABC transporters, biosynthesis of amino acids, two-component system and carbon metabolism, involved mostly in substrates recognition and transport, signal transduction and gene expression regulation. [Conclusion] The results will be expected to enhance our understanding on fluoranthene metabolic pathway and stress response of the DN1 in future studies, as well as lay a solid theoretical foundation for the research on microbial remediation of environmental pollutants.

    • Cr(VI) detoxification characteristics of salt-tolerant Staphylococcus sp. YZ-1 and Bacillus cereus CC-1

      2020, 47(1):66-75. DOI: 10.13344/j.microbiol.china.190358

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      Abstract:[Background] Cr(VI) reducing bacteria are important for bioremediation of high salinity Cr(VI) containing wastewater. However, Cr(VI) detoxification characteristics of salt-tolerant bacteria are little known. [Objective] Comparing the Cr(VI) removal performance and resistant mechanism of two salt-tolerant strains. Identifying the putative Cr(VI) resistance related genes from the result of genome sequencing. Constructing the microbial consortium of Cr(VI) reducing bacteria, and investigate the synergy effect on pollutant removal. [Methods] Staphylococcus sp. YZ-1 was isolated from Chaka salt lake in Qinghai province, and its Cr(VI) removal performance was compared with a previously identified strain Bacillus cereus CC-1. Genome sequencing was used to identify the putative Cr(VI) resistance related genes. [Results] Staphylococcus sp. YZ-1 and Bacillus cereus CC-1 both had Cr(VI) removal characteristics, but the latter was superior. When the Cr(VI) concentration was 0.1 mmol/L, Bacillus cereus CC-1 removed 95.3% Cr(VI) but Staphylococcus sp. YZ-1 only 40.1% in 12 hours. Strain YZ-1 reduced Cr(VI) to organic Cr(III) species, whereas CC-1 removed Cr(VI) through reduction and absorption. Genes encoding pump protein and NAD(P)H oxidoreductase were found in the genome of YZ-1, whereas genes encoding chromate transporter protein ChrA and cytochrome c oxidoreductase were found in the genome of CC-1. The mixture cultures of these strains could self-flocculate and carried the reduced Te0 as participation. The Cr(VI) reductase of YZ-1 was inducible enzyme, and the Cr(VI) reductase of CC-1 was constitutive enzyme. The related encoding genes of multiple Cr(VI) resistance mechanisms maid simultaneously in bacteria. [Conclusion] Staphylococcus sp. YZ-1 and Bacillus cereus CC-1 both are salt-tolerant Cr(VI) reducing bacteria. After mix culture of the two bacteria, the auto-aggregation and Te(IV) reducing performance of YZ-1 may extent the application of Cr(VI) resistant consortium in wastewater treatment.

    • Isolation and classification of Bacillus sp. strains to mine genes encoding perhydrolysis activity

      2020, 47(1):76-87. DOI: 10.13344/j.microbiol.china.190592

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      Abstract:[Background] Subtilisin carlsberg, acetyl xylan esterase and cephalosporin acetyl hydrolase from Bacillus sp. display high perhydrolysis activity in previous reports, and are worth developing for commercial application. [Objective] To produce perhydrolase for enzymatic synthesis of peroxyacetic acid in the future, series of hydrolase with promising perhydrolysis activity-encoding genes were cloned from Bacillus sp. strains isolated from rhizosphere soil samples and natto soybean products. [Methods] Protease-producing thermotolerant Bacillus sp. strains were screened from rhizosphere soil samples and natto soybean products using directed-screening medium, and then identified using 16S rRNA gene sequencing. Three full-length hydrolase genes (including subtilisin carlsberg, acetyl xylan esterase and cephalosporin acetyl hydrolase) were cloned from Bacillus sp. strains and then analyzed using sequence alignment. [Results] Total 85 protease-producing pseudo-Bacillus sp. strains was screened, and then identified and classified into four groups: Bacillus subtilis, Bacillus cereus, Bacillus pumilus and Bacillus megaterium. Three full-length hydrolase genes, subtilisin carlsberg gene (encoding 381 aa) from B. subtilis NSYT-3, acetyl xylan esterase gene (encoding 320 aa) from B. pumilus OSLJ-13, and cephalosporin acetyl hydrolase gene (encoding 318 aa) from B. subtilis NSYT-3, were cloned. All of the simulated 3D structure of subtilisin carlsberg, acetyl xylan esterase, and cephalosporin acetyl hydrolase were coincident with the typical structural characterization of α/β hydrolase fold enzymes. [Conclusion] Cloning and expression of hydrolase with perhydrolysis activity-encoding genes from Bacillus sp. strains will lay the foundation for the enzymatic synthesis technology of peroxyacetic acid.

    • >Fundamentals of Microbiology
    • Effects of knocking-out histone deacetylase gene hdac on cellulase expression in Trichoderma reesei

      2020, 47(1):88-96. DOI: 10.13344/j.microbiol.china.190260

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      Abstract:[Background] Trichoderma reesei is the most representative fungus to produce cellulase. Epigenetic regulation is a heritable change that does not involve changing DNA sequence. Histone deacetylation is one of epigenetic regulation, and histone deacetylase (HDAC) is responsible for deacetylation. Knocking out the acetylase gene can cause a series of changes in spores, hyphae and cellulase activities. [Objective] A mutant of T. reesei Δhdac was developed by knocking out the histone deacetylase (hdac) gene to investigate the effects of hdac on regulation of cellulase expression. [Methods] The hdac knocking-out expression boxes were constructed by the Split-Maker technique, and were transformed into Trichodermium reesei QM9414. After being verified by PCR and Southern blotting, filter paper enzyme activity and carboxymethyl cellulose natriuretic enzyme activity of the mutant were continuously detected for 7 days, and the related gene expressions were detected by RT-qPCR. [Results] The two enzyme activities in mutant strains were 8.00 IU/mL and 30.00 IU/mL higher than those of the start strain Trichodermium reesei QM9414 respectively. The transcription level of cbh1, egl1 and xyr1 in the T. reesei Δhdac were 6.50 times, 6.01 times and 4.51 times higher than those of the start strain, respectively. [Conclusion] The cellulase gene expression in Trichoderma reesei QM9414 was significantly regulated by the histone deacetylase, this provides a new way to study the effects of epigenetic regulation of Trichoderma reesei on cellulase.

    • >Agricultural Microbiology
    • Effect of Streptomyces alfalfae XY25T application on physicochemical properties and microflora in clubroot-diseased soil

      2020, 47(1):97-108. DOI: 10.13344/j.microbiol.china.190244

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      Abstract:[Background] Clubroot, one of the most important soil-borne diseases in the world, is posing a serious threat to the vegetable farming in the Northwest of Hubei, leading to an incidence rate as high as 60% for Chinese cabbage in many fields. [Objective] To study the effects of Streptomyces alfalfae XY25T application on physicochemical properties and microbial community in rhizosphere soil. [Methods] Pot experiments of Chinese cabbage were done in greenhouse, using the diseased soil with S. alfalfae XY25T application as the treatment group and the diseased soil without S. alfalfae XY25T application as the control group. On day 7, 14, 21, 28 and 35 after seedling transplantation, soil samples were collected for analysis of the variations in the enzyme activity and nutrient content in the rhizosphere soil. On day 35, the total DNA was extracted from soil samples. The composition and diversity of bacterial and fungal communities were investigated by using high-throughput sequencing technology combined with bioinformatics analysis. [Results] Compared with the control, the application of S. alfalfae XY25T increased pH value, content of organic matter, alkali-hydrolysable nitrogen, available kalium, and available phosphorus in the soil by 10.0%, 13.7%, 12.0%, 41.1%, and 8.71%, respectively. It also increased the soil enzyme activity, with an increase of 333%, 204%, and 36.1%, 142% in the soil invertase activity, urease activity, catalase activity, and alkaline phosphatase activity. Moreover, S. alfalfae XY25T application improved the soil microbial community structure and diversity, leading to an increase of 54.1%, 22.8%, and 10.7% in the relative abundance of Ascomycota, Zygomycota, and Actinobacteria, in contrast to a 7.59% decrease in the relative abundance of Acidobacteria. Furthermore, S. alfalfae XY25T application also increased the relative abundance of Penicillium, Mortierella, Mucor, and Streptomyces by 25.5%, 14.6%, 8.20%, and 7.24%, respectively. [Conclusion] The application of S. alfalfae XY25T not only improved the soil quality but also significantly altered the microbial community structure and diversity in rhizosphere soil.

    • CRISPR-Cas9 ribonucleoprotein-mediated gene knock-out in Colletotrichum gloeosporioides from Hevea brasiliensis

      2020, 47(1):109-117. DOI: 10.13344/j.microbiol.china.190263

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      Abstract:[Background] Genome editing with CRISPR-Cas9 technique may provide some new ways for gene knock-out, knock-in and gene editing in fungal pathogens. [Objective] To construct a system for gene editing in Colletotrichum gloeosporioides from Hevea brasiliensis. [Methods] Cas9 containing the nuclear localization signal peptide was expressed in Escherichia coli and purified. Potential cleavage sites of URA5 was analyzed and an SgRNA was transcribed in vitro. The Cas9 protein and SgRNA were assembled in vitro to form a ribonucleoprotein. The ribonucleoprotein was transformed into the protoplast of C. gloeosporioides and the transformants were screened. [Results] The Cas9 protein and SgRNA could form a stable ribonucleoprotein and this complex showed high DNA cleavage activity in vitro. The ribonucleoprotein was transformed into the protoplast of C. gloeosporioides and URA5 was successfully knocked out. [Conclusion] The system is convenient for gene editing in C. gloeosporioides of H. brasiliensis.

    • Screening, identification and fermentation optimization of an antimicrobial actinomycete strain 17-7 to Phellinus noxius

      2020, 47(1):118-129. DOI: 10.13344/j.microbiol.china.190160

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      Abstract:[Background] Rubber brown root disease, caused by Phellinus noxius Corner, was a disease causing severely damage to rubber trees and huge economic losses in rubber industry. [Objective] To isolate and screen antimicrobial actinomycetes strains with inhibitory activity on Phellinus noxius from the rhizosphere soil of rubber tree. [Methods] The method of dilution plate coating was applied to isolate actinomycetes; The methods of growth plate-confrontation and mycelia growth inhibition were used to screen antimicrobial actinomycetes. Strain 17-7 was identified based on morphological, physiological, biochemical characteristics and 16S rRNA gene sequence. The conditions of fermentation and medium composition were optimized through single factor and orthogonal experiment. [Results] Strain 17-7 showed antimicrobial activity to five plant pathogens of rubber trees. According to the results of morphological, physiological, biochemical characteristics and 16S rRNA gene sequence, strain 17-7 was identified as Streptomyces samsunensi. The optimum culture conditions of strain 17-7 were with a medium of glucose 20.0 g/L, soy powder 25.0 g/L, KH2PO4 1.0 g/L, NaCl 0.5 g/L, CaCO3 0.5 g/L, and liquid volume 150 mL in 500 mL flask, pH 8.0, rotation speed 140 r/min, inoculation amount 10%, culture temperature at 28 °C for 5 d. [Conclusion] Strain 17-7 was identified as Streptomyces samsunensi and showed strongly inhibitory activity against Phellinus noxius.

    • Isolation and identification of bacteria associated with Anabaena from upland soils in northeast China

      2020, 47(1):130-139. DOI: 10.13344/j.microbiol.china.190209

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      Abstract:[Background] Anabaena distribute widely in agricultural soils and have the ability of fixing-carbon and fixing-nitrogen. Clarifying the relationship between cyanobacteria and their associated bacteria is beneficial to improve the function of Anabaena in agricultural soils. [Objective] To isolate associated bacteria with Anabaena sp. PCC7120 from different upland soils in northeast China, to taxonomically identify the associated bacteria, to speculate the function of associated bacteria, and to provide data support for clarifying the relationship between soil cyanobacteria and their associated bacteria. [Methods] Isolation from cultured plate, PCR-DGGE and cloning-sequencing were used to obtain and analyze 16S rRNA gene sequences of associated bacteria with Anabaena sp. PCC7120 from different upland soils and to determine their taxonomic position. [Results] PCR-DGGE profiles showed there are different quantity and type of associated bacteria with Anabaena sp. PCC7120 from 14 upland soils in northeast China. PCR cloning-sequencing result showed that 37 sequences of associated bacteria were obtained, and 36 sequences were identified at species level. The associated bacteria mainly belong to Sphingopyxis, Variovorax, Flavobacterium, Rhodococcus, and these bacteria were suspected with the functions of adapting oligotrophic condition, enriching trace elements, removing toxins. [Conclusion] There are different quantity and type of associated bacteria with Anabaena sp. PCC7120 from upland soils in northeast China. These associated bacteria were mainly members of Sphingopyxis, Variovorax, Flavobacterium and Rhodococcus.

    • Effects of microbial agents on nutrient and bacterial community diversity in rhizosphere soil of eggplant cultivated in facilities

      2020, 47(1):140-150. DOI: 10.13344/j.microbiol.china.190275

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      Abstract:[Background] Continuous cropping and excessive application of chemical fertilizers of eggplant in greenhouse cause a series of soil quality problems such as imbalance of soil nutrients, decrease of microbial diversity and serious soil-borne diseases. The application of microbial agents can improve soil environmental quality. [Objective] To investigate the effect of microbial agents application on the soil nutrients and bacterial community diversity of eggplant planting soil. [Methods] In the Dahe Experimental Station of Hebei Academy of Agricultural and Forestry Sciences, the microbial agents containing Trichoderma harzianum and Bacillus megaterium were used as fertilizers for eggplant planting in greenhouse. The soil nutrient were detected by traditional chemical methods. The number of culturable microorganisms in soil was measured by dilution plate method and the bacterial diversity and community distribution in rhizosphere were determined by high-throughput sequencing on PCR-amplified 16S rRNA gene V3?V4 fragments. [Results] Compared with control group, microbial agents treatment increased the nutrients of rhizosphere soil in eggplant flowering and seedling-pulling stages. The total nitrogen, alkali-hydrolyzed nitrogen, available phosphorus and available potassium in the soil of microbial agents treatment at seedling-pulling stage increased by 13.85%, 21.07%, 31.51% and 55.94% respectively compared with that of control. Application of microbial agents changed the quantity and composition of culturable microorganisms in soil. Microbial agents increased the number of bacteria, fungi, actinomycetes and the proportion of bacteria and actinomycetes in the total microbial number. The Shannon index was higher and the Simpson index was lower in microbial agents treatment than that in control group. However, ACE index and Chao1 index had no significant difference between microbial agents treatment and control. The results indicated that microbial agents can increase the diversity of soil microbial communities, and have no significant impact on microbial community richness. Analysis of microbial communities of soil treated with microbial agents or not showed that microbial agents treatment increased the content of Xanthomonadales, Rhodospirillales, Sphingobacteriales, Bacillales, Cytophagales, Pseudomonadale and decreased the content of Burholderiales and Flavobacteriales in soil. The application of microbial agents could promote the vegetative growth of eggplant and increase the yield of eggplant by 18.52%. [Conclusion] As a new type of environmental protection fertilizer, microbial agents can improve soil nutrition, increase the number of culturable microorganisms and improve soil microbial diversity. The results provided a scientific basis for the rational application of microbial agents.

    • >Food Microbiology
    • Flavor-related microbiota and their flavor metabolism during highland barley Baijiu fermentation

      2020, 47(1):151-161. DOI: 10.13344/j.microbiol.china.190240

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      Abstract:[Background] Highland barley Baijiu is a product of multispecies solid-state fermentation. It is important to regulate the fermentation of highland barley Baijiu through analyzing the important functional microbiota and their metabolic characteristics. [Objective] The purpose of this study was to reveal the flavor-related microbiota during highland barley Baijiu fermentation and resolve their co-metabolism. [Methods] the diversity and composition of microbial community during highland barley Baijiu fermentation were revealed by high-throughput sequencing. the flavors of fermented grains were detected by HS-SPME-GC-MS. Then flavor-related microbiota during highland barley Baijiu fermentation was revealed by correlation analysis between microbes and flavors. The effect of physical and chemical factors to the flavor-related microbiota was revealed by the Mantel test. the simulated fermentation with six strains under laboratory conditions was carried out to resolve their co-metabolism. [Results] nine dominant fungus and eight dominant bacteria (relative abundance>1%) were found during highland barley Baijiu fermentation. Aspergillus, Komagataella, Lactobacillus, Pichia, Saccharomyces and Weissella were identified as flavor-related microbiota. Reducing sugar (r2=0.946 9, P=0.013 2) and acidity (r2=0.847 6, P=0.048 6) were determined as key factors driving the succession of the flavor-related microbiota. The simulated fermentation with six strains revealed the in vitro system had the same microbial succession and flavors structure as the in situ system. [Conclusion] This work revealed the flavor-related microbiota and key factors driving their succession, and verified the metabolic characteristics of flavor-related microbiota by simulated fermentation. These results might provide a new perspective for regulating flavors information during highland barley Baijiu fermentation.

    • >Veterinary Microbiology
    • Biofilm-forming capability and drug resistance of extraintestinal pathogenic Escherichia coli isolated from diseased swine

      2020, 47(1):162-171. DOI: 10.13344/j.microbiol.china.190355

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      Abstract:[Background] Porcine extraintestinal pathogenic Escherichia coli (ExPEc), a pathogen seriously endangers the swine industry. However, few studies have reported the biofilm-forming capability and drug resistance of porcine ExPEc. [Objective] To study the relationship between the biofilm-forming capability and drug resistance of 3 ExPEc strains isolated from the diseased swine lung, and provide the reference to prevent and treat the porcine ExPEc disease in the perspective of anti-biofilm formation. [Methods] The optimal conditions and capability for biofilm formation of porcine ExPEc strains were examined using 96-well plate crystal violet staining combined with orthogonal experiments. The morphologic features of the biofilms produced by each strain were observed using a scanning electron microscopy. Biofilm-forming related genes carried by each strain were detected by PCR, as well as the minimum inhibitory concentration (MIC) of antibiotic to porcine ExPEc strains both in floating form and biofilm form were determined by micro-broth dilution method, respectively. [Results] The optimal film-forming conditions of 3 ExPEc strains were not consistent, but showed strong biofilm formation under their optimum conditions, and carried synchronously 10 kinds of biofilm-forming related genes (pgaA, pgaB, pgaC, pgaD, luxS, fimA, hipA, iha, flhC, flhD). Scanning electron microscopic observation showed that the strain SE-1 aggregated to form a flaky biofilm, while both SE-2 and SE-3 strains aggregated to form multi-layered biofilms, and there was obvious space between aggregates. The MIC values of some tested antibiotics to 3 strains growing in a biofilm were 2?32 times higher than those of corresponding planktonic bacteria, and the drug sensitivity changed from sensitivity to medium or resistance. [Conclusion] The result in the study provides a basis for the prevention and treatment of porcine ExPEc infection from the perspective of anti-biofilm formation.

    • >Aquatic Microbiology
    • Effects of light intensity and nitrogen concentration on the growth and biochemical composition of filamentous green alga Zygnema sp.

      2020, 47(1):172-181. DOI: 10.13344/j.microbiol.china.190171

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      Abstract:[Background] Environmental factors and nutrient factors have significant effects on the growth and biochemical composition of microalgae, and light intensity and nitrogen concentration are two of important environmental factors. [Objective] The effects of different light intensities and initial nitrogen concentrations on the growth and biochemical composition of filamentous green alga, Zygnema sp. was investigated. [Methods] The alga was grown in bubble column glass photobioreactor with modified BBM medium under two light intensities (100 μmol/(m2·s) and 300 μmol/(m2·s)) and six initial nitrogen concentration gradients (3, 6, 9, 12, 15 and 18 mmol/L). [Results] The highest biomass was observed at high light intensity (300 μmol/(m2·s)) and 12 mmol/L initial nitrogen concentration, which was 6.60 g/L. The initial low nitrogen concentration (3 mmol/L) was conducive to the accumulation of total lipid and fatty acids of Zygnema sp., and the highest total lipid content reached 32.13% of dry weight. The main components of fatty acid were palmic acid, oleic acid, linoleic acid and α-linolenic acid, and oleic acid content accounted for 55.01% of total fatty acid. Under low light intensity (100 μmol/(m2·s)), the highest protein and carbohydrate content was obtained under 18 mmol/L initial nitrogen concentration, which was 16.35% of dry weight and 37.70% of dry weight, respectively. Meanwhile, the total lipid content accounted for only 10.16% of dry weight. [Conclusion] The light intensity and initial nitrogen concentration had a great influence on the growth and biochemical composition of Zygnema sp., the target accumulation of metabolites of Zygnema sp. could be effectively increased by adjust the light intensity and initial nitrogen concentration.

    • Polyclonal antibody preparation and application of grass carp reovirus genotype III nonstructural protein NS66

      2020, 47(1):182-189. DOI: 10.13344/j.microbiol.china.190479

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      Abstract:[Background] Grass carp reovirus (GCRV) 104 strain can cause typical grass carp hemorrhagic disease, the analysis of its coding fragments is helpful to provide a basis for clinical immunological detection. [Objective] To study the possible function of the NS66 protein encoded by the s6 gene segment of GCRV 104 strain, the highly specific polyclonal antibody against NS66 protein was prepared, and its specificity was identified. [Methods] s6 gene segment amplified by PCR was cloned into the expression vector pGEX-4T-3, transformed into E. coli BL21 and induced by IPTG. After analysis and identification by SDS-PAGE, the target protein is obtained through purification. Mice were then immunized with purified recombinant protein to obtain polyclonal antibody. Titers were determined by Western blot, and antibody specificity was identified by Western blot and IFA (indirect immunofluorescence assay). [Results] The recombinant protein analysed by SDS-PAGE was about 66 kD, the same size as expected. The target protein was mainly present in inclusion bodies. The polyclonal antibody titer prepared by Western blot was more than 1:50 000. Western blot and IFA showed that the prepared polyclonal antibody recognizes the 104 strain, and the polyclonal antibody recognizes a single band with high specificity. [Conclusion] The study showed that the polyclonal antibody against NS66 protein can specifically recognize GCRV104 virus, which lays a foundation for the establishment of GCRV104 immunodiagnostic method and the study of GCRV-encoded NS66 protein.

    • >Microbial Engineering and Medicine
    • Specificity of the loading acyltransferase from avermectin modular polyketide synthase

      2020, 47(1):190-199. DOI: 10.13344/j.microbiol.china.190236

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      Abstract:[Background] Under normal conditions in vivo, loading acyltransferase from avermectin modular polyketide synthase (AveAT0) recruits 2-methylbutyryl-coenzyme A (CoA) or isobutyryl-CoA as starter units to synthesize avermectins of “a” series or “b” series, respectively. [Objective] We are aiming to explore the catalytic specificity of AveAT0 on 2-methylbutyry-SNAC and isobutyryl-SNAC, as substrate analogues of 2-methylbutyryl-CoA and isobutyryl-CoA and modify its favorability towards the former. [Methods] The protein sequences of several AT0s were compared and residues playing important roles in protein-substrate identification were uncovered. The mutation experiments were then conducted on AveAT0 and concentration of free sulfhydryl (SH) released from SNAC was obtained via Ellman test to characterize the substrate speci?city of mutated enzymes. [Results] The Km value of AveAT0 towards 2-methylbutyry-SNAC was 0.4 mmol/L, kcat value was 14.1 min–1 and kcat/Km value was 32.1 L/(mmol·min); the Km value of AveAT0 towards isobutyryl-SNAC was 0.8 mmol/L, kcat value was 6.4 min–1 and the kcat/Km value was 7.5 L/(mmol·min). The mutation sites were selected as V224M, Q149L and L121M. After trials in combined mutations, the specificity of a triple mutant AveAT0 V224M/Q149L/L121M for both substrates increased. The Km value of AveAT0 V224M/Q149L/L121M towards 2-methylbutyryl SNAC was 0.8 mmol/L, kcat value was 5.4 min–1, and kcat/Km value was 6.9 L/(mmol·min); the kcat/Km towards isobutyryl-SNAC was 0.1 L/(mmol·min). [Conclusion] This study found key residues in AveAT0 identification, and provided insight for the rational modification of avermectin polyketide synthase acyltransferase.

    • >Pharmaceutical Microbiology
    • High throughput screening and combined breeding of Streptomyces lomondensis for high production of lomofungin

      2020, 47(1):200-209. DOI: 10.13344/j.microbiol.china.190297

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      Abstract:[Background] Lomofungin, a phenazine compound with broad-spectrum antibacterial activity, can be biosynthesized by Streptomyces lomondensis S015. [Objective] Due to the low production of lomofungin in S015, this study aims to enhance its production by combined mutation breeding and genetic engineering. [Methods] A high throughput screening method was established to analyze the production of lomofungin of mutants, and the lomofungin high-production strains were screened after atmospheric and room temperature plasma (ARTP) technology and ultraviolet (UV) combined mutation breeding based on the starting strain S015. Then, trpE1 and trpE2 genes which are key genes in chorismate-competing pathway were knocked out in the lomofungin high-production mutant, and the global regulatory gene afsR was overexpressed. [Results] According to lomofungin’s characterized UV absorption peak at 375?nm and the positive correlation between the concentration of lomofungin and the value of A375, a high throughput screening method, which is based on 24-deep-well plate culture and microplate reader, was built successfully. After 6 rounds of ARTP and UV combined mutation breeding, strain M6, a genetically stable and high lomofungin-production strain, was screened from 4 320 mutants. Its yield of lomofungin is 61.33 mg/L, which is 7.35 times the yield of starting strain S015. After two branch pathway genes trpE1 and trpE2 were knocked out from strain M6, the lomofungin production increased to 81.89?mg/L, and then it increased to 109.53 mg/L when afsR was overexpressed, which is 13.13 times the production of S015. [Conclusion] The high throughput screening method developed in this study could efficiently analyze the production of lomofungin of mutants in a simple and fast way. Combined mutation breeding of ARTP and UV as well as genetic engineering is an effective mean to obtain high-yield strains of antibiotics.

    • Biological characteristics and complete genomic analysis of a novel virulent bacteriophage that infects Klebsiella pneumoniae capsular type K63

      2020, 47(1):210-221. DOI: 10.13344/j.microbiol.china.190585

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      Abstract:[Background] Bacteriophages have a specific bactericidal ability to influence the evolution of bacteria and ecology. In recent years, due to the global emergence of multidrug-resistant bacterial strains, phage therapy has received renewed interest. [Objective] Biological characteristics and complete genome sequence of a novel virulent bacteriophage vB_KpnP_IME308 that infects Klebsiella pneumoniae capsular type K63. [Methods] A novel lytic phage was isolated from sewage of Chinese general Hospital of the PLA using a clinical Klebsiella pneumoniae strain as an indicator. The double-layer plate method was conducted the titer, optimal multiplicity of infection (MOI), one-step growth curve, thermostability and pH stability. Phage morphology was observed by transmission electron microscopy after purification. Phage genome was sequenced using the Illumina Miseq sequencing platform. Complete genome sequence was used for genome annotation, comparative genomics and evolutionary analyses. [Results] A novel phage, designated vB_KpnP_IME308, isolated from hospital sewage using a clinical Klebsiella pneumoniae strain as a host. The optimal MOI of phage vB_KpnP_IME308 is 0.001. One step growth curve showed that the latent period and the burst period of vB_KpnP_IME308 were 20 min and 80 min, respectively. The burst size was about 330 PFU/cell. The genome of vB_KpnP_IME308 is 43 091 base pairs (bp) with (G+C)mol% content of 59.3% and (A+T)mol% content of 46.1%. Electron microscopic observation showed that the phage belongs to the family Podoviridae. The BLASTn alignment showed that the genome of the phage had limited similarity with the currently known phages. The evolutionary relationship between phage vB_KpnP_IME308 and other Podoviridae phages was analyzed by major capsid protein and large terminase protein of phage vB_KpnP_IME308, which suggest that phage vB_KpnP_IME308 was a member of the genus Drulisvirus of subfamily Autographivirinae. [Conclusion] A lytic phage, vB_KpnP_IME308, was isolated successfully from hospital sewage, characterization and genome analysis of a novel bacteriophage vB_KpnP_IME308, which shows its potency to be developed as a novel alternative for multi-drug resistant Klebsiella pneumoniae infects control and treatment.

    • >REVIEWS
    • Emission and potential risks of bioaerosols in sanitary landfill

      2020, 47(1):222-233. DOI: 10.13344/j.microbiol.china.190527

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      Abstract:With the improvement of the bioaerosol understanding, more and more attention has been paid to their sources, pollution characteristics and health risks. Sanitary landfill is one of the main sources of bioaerosol. This paper explains the concentration level, particle size distribution and microbial population structure of microorganisms in aerosol particles of sanitary landfill, analyses the pollution characteristics, influencing factors and potential risks, and prospects the future hot spot and the main direction of microbial emissions of sanitary landfill. This paper will supply scientific basis and reference for the control and reduction of bioaerosols in sanitary landfill.

    • Progress in fatty acids production by thraustochytrids using industrial and agricultural wastes

      2020, 47(1):234-243. DOI: 10.13344/j.microbiol.china.190474

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      Abstract:Thraustochytrids produce fatty acids in high yields. However, the high cost of raw materials required by the traditional culture method has largely hindered the industrialization of thraustochytrids. Searching for cheap raw materials with high utility and easy availability has thus become a major research concern. In this paper, thraustochytrids and their potential in producing fatty acids are reviewed, with a focus on the utilization of industrial and agricultural wastes as nutritional sources in the production of fatty acids by thraustochytrids. We expect this review to provide information for future research.

    • A new immunization strategy against Chlamydia trachomatis infection in female genital tract

      2020, 47(1):244-252. DOI: 10.13344/j.microbiol.china.190183

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      Abstract:Chlamydia trachomatis is the main pathogen of trachoma and genitourinary infections. According to the World Health Organization (2015) statistics, there are about 130 million new cases of Chlamydia trachomatis infection every year in the world. Studies have shown that CD4+Th1 cellular immune response plays?an important role in resistance to Chlamydia trachomatis infection. Therefore, based on the immune response characteristics of anti-Chlamydia trachomatis infection, the researchers constructed many candidate vaccines, but none of them were successfully applied in clinic. In recent years, it has been found that there are not only humoral immunity and cellular immunity, but also some noticeable immune cells resident in mucosa of reproductive tract, suggesting that enhancing mucosa immunity can be used as a potential way to prevent Chlamydia trachomatis infection. It is a new immunization strategy against Chlamydia trachomatis infection in reproductive tract. In this paper, the progress of mucosa immunization and Chlamydia trachomatis infection in female reproductive tract is reviewed, and some suggestions for the development of Chlamydia trachomatis vaccine in the future are provided.

    • New progress of protein glycosylation modification in gut microbes

      2020, 47(1):253-262. DOI: 10.13344/j.microbiol.china.190309

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      Abstract:Gut microbes play an important role in maintaining human health and inducing disease development. The glycosylation modification of gut microbes has a profound impact on the host’s life activities. From the perspective of glycomics, this review discusses and analyzes the composition and function of gut microbes, the glycosylation patterns of representative bacteria and their closely related physiological functions. We aslo summarize the regulation ways of glycosylation on gut microbial functions and activities.

    • Research progress in enteroids in porcine enteric coronavirus infection

      2020, 47(1):263-271. DOI: 10.13344/j.microbiol.china.190343

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      Abstract:Porcine enteric coronavirus, an important agent of diarrhea in piglets, primarily infects the villous epithelia of the small intestine, causing substantial economic losses to the pork industry. The lack of an in vitro model that can recapitulate the highly complex physiological properties of the gastrointestinal tract significantly limits the study of the interactions between porcine enteric coronavirus infection and host intestinal epithelium. With the rapid development of stem cell technology, an in vitro model that can mimic diverse cellular nature and complex structure of the intestine-enteroids, has attracted widespread attention. Compared with conventional cell lines, enteroids not only simulate the structure and function of the intestine, but also retain the genetic characteristics of the host. Enteroids would be expected to be an ideal model for studying the interactions of host-enteric pathogens. This article reviews the recent research progress of porcine enteric coronavirus and the applications of enteroids in enteric pathogens research, in order to provide new insights for the future fundamental research of porcine enteric coronavirus.

    • Cell-cell communication in human fungal pathogen Cryptococcus neoformans

      2020, 47(1):272-281. DOI: 10.13344/j.microbiol.china.190598

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      Abstract:Cell-cell communication has been well known about the control of various fundamental processes, such as proliferation and cellular differentiation, in multi-cellular organisms. In microbes, cell-cell communication system determines a variety of community and social behaviors. Cryptococcus neoformans is an important environmental human fungal pathogen, which is a common cause for fatal infection towards immune-compromised patients. This pathogen employs diverse strategies as environmental adaptation to maximize its survival during or between its infections. These strategies include cell-cell communication, which regulates the transition between different Cryptococcus neoformans morphotypes with distinct trade-offs against stressors from host or natural niches. In this review, the complex cell-cell communication systems in C. neoformans and their contribution to morphotype transition, stress adaptation and community behaviors are focused.

    • Research progress on microbial degradation of dinitroaniline herbicides

      2020, 47(1):282-294. DOI: 10.13344/j.microbiol.china.190213

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      Abstract:Dinitroaniline herbicides are a class of highly efficient pre-emergence herbicides, with broad spectrum and wide application. Microbial catabolism plays the most important role in the dissipation of the dinitroanilines in the environment. Isolating highly efficient herbicides-degrading microorganisms, studying the metabolic pathways, and elucidating the degradation mechanisms will provide the theoretical basis and degrading resources for the study on the transformation mechanism and ecological security of the herbicides in the environment, and for the microbial remediation of the herbicides residual contamination in the environment. This paper summarizes the research progress on microbial degradation of dinitroaniline herbicides, including degrading bacteria, metabolic pathways, and the genes/enzymes involved in the degrading progress. The aim of this review is to provide theoretical basis and resources for the bioremediation of dinitroaniline herbicides residual contamination in environment.

    • Microbial origin of natural products isolated from ascidians

      2020, 47(1):295-304. DOI: 10.13344/j.microbiol.china.190436

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      Abstract:Marine animals are important sources of marine natural products with biological activity. Ascidians harbor rich microbial communities, including bacteria, actinobacteria, fungi, and cyanobacteria. More and more direct or indirect evidence suggests that some natural products isolated from ascidians are not produced by ascidian themselves, but by their symbiotic microorganisms. In this review, we present the research methods of microbial origin of ascidians natural products in recent years, including direct methods, such as the isolation of culturable bacteria, crude extract detection of non-culturable bacteria, metagenomics, whole genome sequencing, as well as indirect methods of the comparison of compound structures. The study of biosynthetic origin of natural products isolated from marine microorganism-ascidians assemblages, can solve problem of crude drug from animal sources, and provide the evidence for symbiotic relationship between ascidians and microorganisms.

    • >EDUCATION
    • Cultivating talent with wide vision: core curriculum construction in Microbiology

      2020, 47(1):305-310. DOI: 10.13344/j.microbiol.china.190437

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      Abstract:Microbiology, as a basic course in biology, plays a vital role in building students’ knowledge systems and teaching values in classroom. In order to achieve the goal of “golden class” construction, the microbiology course adheres to the solid foundation and pays attention to the cutting-edge concept in the construction process. It emphasizes the connection and transition of different courses in the curriculum designing, and provides special lectures. Besides, we built high-quality teaching materials, apply the platform of “duifene”, and carry out activities such as “I am the keynote speaker” to enhance the quality of teaching and extend the teaching effect. At the same time, we also have a deep understanding of talent training, not only to cultivate intelligence, but also to educate people, to combine professional content in teaching, to stimulate students’ sense of social responsibility and mission, to give students the goal and guidance of learning by role models, to strengthen moral education, and then achieve the goal of all-round education.

    • >BIOLOGICAL LAB
    • Development and application of a multiplex real-time PCR assay for quantitative detection of three pathogens related to kiwifruit root rot disease

      2020, 47(1):311-321. DOI: 10.13344/j.microbiol.china.190410

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      Abstract:[Background] In recent years, with the expansion of kiwifruit cultivation area, frequent occurrences of diseases have increasingly affected the yield and quality of kiwifruit. Phytophthora cactorum, P. cinnamomi and P. lateralis are a group of pathogens that cause the kiwifruit root rot disease. [Objective] The present study aimed to establish and optimize a multiplex quantitative real-time PCR assay for simultaneously detecting the three pathogenic Phytophthora species, and to investigate the distribution of these pathogens in the main production areas of kiwifruit. [Methods] The Ypt1 (ras-related protein gene) sequences were aligned to develop species-specific TaqMan probes and primers for P. cactorum, P. cinnamomi and P. lateralis, respectively. A multiplex quantitative real-time PCR assay was established and optimized, and the specificity and sensitivity were tested. Finally, the detection system was used to analyze the Ypt1 gene content of three pathogens from the rhizosphere soils in the main production areas of kiwifruit. [Results] HEX, FAM and ROX fluorescence signals were detected in the DNA samples of P. cactorum, P. cinnamomi and P. lateralis, respectively, but no fluorescence signals in those of their closely related and other soil-borne pathogens. The sensitivity was 100 fg for each pathogen. By assaying 166 rhizosphere soil samples of kiwifruit plants from Zhouzhi and Meixian Prefecture of Shaanxi Province, P. cactorum was found to be the most widely distributed along with the highest Ypt1 gene content, while P. cinnamomi and P. lateralis were relatively less frequent. [Conclusion] The established multiplex quantitative detection method was specific and sensitive, and was suitable for the detection and quantification of P. cactorum, P. cinnamomi and P. lateralis. This technique would be useful in early diagnosis, monitoring and prevention of Phytophthora disease in kiwifruits.

    • Construction and application of a novel plasmid for promoter activity determination of Brucella genes base on nanoLuc luciferase

      2020, 47(1):322-329. DOI: 10.13344/j.microbiol.china.190141

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      Abstract:[Background] Brucellosis is a zoonotic infectious disease caused by Brucella spp. and threats the development of animal husbandry and human health. A novel plasmid was constructed for promoter activity determination in Brucella based on NanoLuc luciferase gene (nluc), which is important for research on regulatory mechanism of Brucella virulence genes. [Objective] Preparation of rabbit polyclonal antibody of Nluc, construction of a Nluc reporter plasmid for promoter activation determination in Brucella, and verification of the Nluc reporter plasmid for Brucella bcsp31 gene and virB promoter. [Methods] The nluc gene was ligated into prokaryotic expression vector pET-28a and constructed as recombinant vector pET-Nluc. The New Zealand rabbit was immunized to prepare polyclonal antibody of Nluc protein. The plasmids pNluc, pBcsp31-Luc and pVirB-Luc were constructed based on a broad-host-range vector pBBR1MCS. Brucella recombinant strains S2308(Nluc), S2308(Bcsp31-Nluc) and S2308(VirB-Nluc) were constructed by electrotransformation of plasmids. The promoter activity of bcsp31 and virB were detected in TSB. The activity of virB promoter in Brucella was compared in TSB and within RAW264.7 cells. [Results] The Nluc protein was expressed and purified. The ELISA titer of polyclonal antibody was approximately to 1:100 000. The plasmid pNluc, pBcsp31-Luc, pVirB-Luc and the S2308(Nluc), S2308(Bcsp31-Nluc), S2308(VirB-Nluc) strains were constructed successfully. The results of bcsp31 and virB promoter activity in TSB showed that promoter activity can be detected accurately in pNluc plasmid. The result of virB promoter activity in intracellular Brucella showed that the activity of virB promoter is enhanced significantly. [Conclusion] In this study, a plasmid for promoter activity determination of Brucella genes was constructed successfully. The results of this study showed that the promoter activity of Brucella genes can be detected accurately in pNluc plasmid. This study provides a novel strategy for determining promoter activity of Brucella virulence genes, which may benefit investigation of Brucella pathogenesis.

    • Establishment and application of indirect ELISA for detection of bovine coronavirus antibody

      2020, 47(1):330-338. DOI: 10.13344/j.microbiol.china.190422

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      Abstract:[Background] Bovine coronavirus (BCoV) is one of the main causes of neonatal calf death, and effective detection is the prerequisite to prevent and control the disease. [Objective] At present, BCoV ELISA detection method has some defects, such as low sensitivity, instability and so on. This study aims to improve these defections to establish indirect ELISA detection method. [Methods] The method of indirect ELISA was established by using the soluble recombinant N protein of the epidemic BCoV-CD strain as an antigen. The epitope of the N protein was predicted by DNAStar soft, and was prepared by prokaryotic expression in the non-denatured condition. The seroepidemiological investigation of BCoV infection in Heilongjiang province in recent 5 years was carried out by using this method. [Results] The optimum working conditions of the ELISA method were as follows: the coating solution was 50 mmol/L pH 9.6 carbonate, and the antigen coating concentration was 2.5 μg/mL; The sample diluent was PBST, the dilution concentration was 1 μg/mL, and incubated at 37 °C for 1.5 h; The dilution concentration of HRP-labeled secondary antibody was 1:7 500, and incubated at 37 °C for 1.0 h; The blocked condition was 1% gelatin at 37 °C for 30 minutes. The negative-positive cut off value was 0.225. The method had no cross-reaction with positive serum of bovine rotavirus, bovine viral diarrhea virus, bovine respiratory syncytial body, bovine infectious rhinotracheitis, bovine parainfluenza virus type 3 and Escherichia coli. The intra-and inter-assay coefficient of variation was less than 10%, and the coincident rate with virus neutralization test was 93.5%. The results showed that the positive rate of BCoV antibody was 98.84% in 603 serum samples of cows in some areas of Heilongjiang Province. [Conclusion] The ELISA method established in this study has strong specificity, high sensitivity and good stability, which provides a technical basis for the further development of ELISA kit.

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