• Volume 45,Issue 12,2018 Table of Contents
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    • >Microbial Functional Genomics
    • miR-34b regulates the replication of Enterovirus 71 in rhabdomyosarcoma cell

      2018, 45(12):2731-2737. DOI: 10.13344/j.microbiol.china.180401

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      Abstract:[Background] MicroRNAs (miRNAs) play an important role in the process of infection and replication of virus in host cells. [Objective] In this study, we examined miR-34b effect on the replication of Enterovirus 71 in RD (rhabdomyosarcoma) cells. [Methods] Western blot and real-time PCR test were performed to investigate the effect of miRNAs on viral replication. Furthermore, dual-luciferase reporter assay was used to detect the interaction between miR-34b and potential target eIF4E, and the expression of eIF4E at mRNA levels was tested by real-time PCR. [Results] The study showed that miR-34b can stimulate the replication of EV71 virus. On the contrary, miR-34b inhibitor can suppress EV71 virus replication. Cellular miR-34b can regulate the replication of EV71 virus through binding to eIF4E in host cells. [Conclusion] Our paper should report the role of miR-34b in this regulation for the first time. Our findings support the notion that the cellular miRNAs play an important role in the host and virus infection.

    • CRISPR-Cas9-assisting efficient and sequential genome deletions in Mycobacterium smegmatis

      2018, 45(12):2738-2750. DOI: 10.13344/j.microbiol.china.180118

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      Abstract:[Background] For its fast growing and non-pathogenic property, Mycobacterium smegmatis is used as a model strain for pathogenic Mycobacterium tuberculosis and a workhorse to produce steroid hormones. But it lacks efficient genome deletion system. [Objective] In this study, we established a high efficiency genome deletion system in M. smegmatis assisted by CRISPR-Cas9. [Methods] The Cas9 backbone plasmid pCas9101 was constructed with a tetracycline-inducible codon-optimized cas9 expression operon. Approximate 1 kb flanked homologous arms and proper gRNA operon were used to construct vectors to study the deletion efficiency on 3β-hydroxysteroid dehydrogenase (MSMEG_5228, 1 071 bp) encoding gene and cholesterol degradation gene cluster (MSMEG_5990-MSMEG_6043, about 48 kb) in M. smegmatis mc2155. The classical p2NIL-pGOAL method with same homologous arms was used as the control to delete the two same DNA segments in M. smegmatis mc2155. Their deletion efficiency were calculated and compared. [Results] When using CRISPR-Cas9 assisted method, 22% and 18% clones showed the expected deletions in MSMEG_5228 gene and cholesterol-degrading gene cluster, respectively. Even the sequential deletion efficiency on both segments can reach 4%. However, no expected deletion mutants were obtained in our experiments with p2NIL-pGOAL method. [Conclusion] This CRISPR-Cas9 assisted system can facilitate genome deletion in M. smegmatis mc2155, and provide a fast and efficient genome manipulation approach for Mycobacterium in the future.

    • >Microbial Model Strain
    • Phylogenetic analysis of Lactobacillus casei and closely related species/subspecies based on sequences of housekeeping genes

      2018, 45(12):2751-2761. DOI: 10.13344/j.microbiol.china.180105

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      Abstract:[Background] 16S rRNA gene sequence is widely used in the classification and identification of bacteria, although still with some limitations. The housekeeping gene as a molecular marker has its unique advantages in phylogenetic analysis among related species and subspecies. [Objective] We used 16S rRNA, uvrC (excinuclease ABC subunit C) and murE (UDP-N-acetylmuramyl tripeptide synthase) gene sequences to identify the phylogenetic relationship among Lactobacillus casei and its related species/subspecies. [Methods] six L. casei strains isolated from traditional dairy products were selected. The uvrC and murE gene sequences of each isolates were obtained by polymerase chain reaction (PCR) methods and sequencing technology. Then, we estimated the genetic distance and constructed phylogenetic trees of 6 L. casei strains combined with 5 published related species/subspecies based on the sequences of 16S rRNA gene, uvrC and murE gene, respectively. [Results] Phylogenetic trees indicated that the homology of closely related L. casei species among uvrC, murE, the combined genes (uvrC-murE) and 16S rRNA gene were different, ranged from 79.00% to 99.16%, 89.08% to 99.20%, 76.56% to 99.69% and 99.58% to 100%. L. casei and its related species and subspecies cannot be distinguished by 16S rRNA gene sequence analysis. The phylogenetic tree based on the sequences of combined gene (uvrC-murE) had a distinct classification on genetic relationship of L. casei and its related species and subspecies. [Conclusion] Phylogenetic analysis based on the combined gene (uvrC-murE) can be used as a supplementary of 16S rRNA gene analysis for rapid classification and identification of L. casei and its related species and subspecies.

    • >Industrial Microbiology
    • Production of succinate by a metabolic engineered Escherichia coli and its scale-up process in fermentor

      2018, 45(12):2541-2551. DOI: 10.13344/j.microbiol.china.180113

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      Abstract:[Background] Escherichia coli AFP111 produces succinate with low yield from glucose and with high production of acetate. [Objective] To reduce acetate production and improve succinate yield. A 100-L-scale fermentation process was established. [Methods] One-step homologous recombination was used for knocking out the key enzyme gene of acetate synthesis pathway and modifying key enzyme promoter of succinate synthesis pathway. The culture conditions were optimized by using single factor design in a 5-L fermentor. [Results] The resulting strain of SX02, deletion of ackA-pta and tdcDE, reduced acetate production by 53.42%, and increased succinate yield by 9.85% as compared to the original strain. The glucokinase activity of SX03, obtained after over-expressing glk, was 3.66 times high than SX02. Its acetate production reduced by 31.62% and its succinate yield increased by 8.28% as compared to SX02. SX03 produced 3.97 g/L acetate, and 1.62 mol of succinate per mol glucose in a 5-L fermentor with a decrease of acetate production by 75.76% and an increase of succinate yield by 19.12% as compared to the original strain. The fermentation conditions including various neutralizers pH values, agitation speed, glucose concentrations were optimized in a 5-L fermentor. The optimal fermentation conditions were pH 6.8, agitation speed of 250 r/min, glucose concentration of 100 g/L. Under the optimal conditions, SX03 produced 2.24 g/L acetate and 1.66 mol of succinate per mol glucose with a decrease of acetate production by 20.65% and an increase of succinate yield by 2.47%. The fermentation using the strain SX03 was scaled up in a 100-L fermentor, which produced 1.91 g/L acetate and 1.30 mol of succinate per mol glucose. [Conclusion] The metabolic engineered Escherichia coli shows great industrial potential for the commercial production of succinate.

    • Inducible heterologous expression and carbon catabolite repression of β-amylase from Bacillus megaterium in Bacillus subtilis

      2018, 45(12):2552-2562. DOI: 10.13344/j.microbiol.china.171059

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      Abstract:[Background] β-Amylases have been widely used in food and medical fields. Most industrial β-amylases are extracted from plants, hampering the application of β-amylase due to high costs. Microbial production of β-amylase has been reported before but has not been industrialized because of the low yields. [Objective] To achieve an efficient inducible expression of a β-amylase from Bacillus megaterium in Bacillus subtilis, relieve carbon catabolite repression (CCR) exerted on the expression of recombinant β-amylase and characterize the recombinant enzyme. [Methods] A xylose-induced vector was constructed to mediate the expression of the amyM gene from Bacillus megaterium 1514 encoding a β-amylase in Bacillus subtilis. CCR of the recombinant β-amylase was studied by site-directed mutagenesis of the catabolite responsive element (CRE) located within the signal peptide-encoding region of amyM. [Results] The recombinant Bacillus subtilis that inductively expressed the β-amylase was obtained. The yield of the recombinant enzyme was significantly improved by silent mutagenesis of conserved nucleotide within amyM-CRE. The recombinant β-amylase had a molecular size of 57 kD and hydrolyzed soluble starch to yield 72% maltose and a little glucose. The enzyme was optimally active at pH 6.0 and 50 °C. Co2+ and Ca2+ increased the efficiency of enzymatic hydrolysis. [Conclusion] Highly efficient expression of β-amylase was achieved to provide experimental support for the industrial production of β-amylase from fermentation.

    • Improvement of acid-tolerance of Lactococcus lactis NZ9000 by aspartate

      2018, 45(12):2563-2575. DOI: 10.13344/j.microbiol.china.180002

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      Abstract:[Background] As an important microorganism in fermentation, lactic acid bacteria are confronted with severe acid stress during the process of production. [Objective] We analyzed the specific mechanism how aspartic acid improved acid stress resistance of L. lactis NZ9000. [Methods] The effects of aspartate on transcription levels of key genes in energy production and amino acid metabolic pathways were analyzed by fluorescence quantitative PCR under acid stress. The content of aspartic acid was increased by overexpression of L-asparaginase. [Results] The main mechanism of aspartic acid to improve the acid stress resistance was generating oxaloacetate and glutamate in the role of transaminase. Oxaloacetate entered three carboxylic acid cycle and can provide more energy for cells; glutamate promoted acid resistance through glutamic acid decarboxylase pathway. The transcription levels of genes of glycolysis and three carboxylic acid cycle pathway were up-regulated and the content of intracellular ATP was 42 times as high as that in the control group at pH 4.0 for some time. The intracellular glutamate content was 1.99 times higher than that of the control. The survival rate of recombinant strain obtained by overexpression L-asparaginase was about 11.11 times as high as that of the control at pH 3.6 for 0.5 h. [Conclusion] Aspartic acid could improve acid stress resistance of L. lactis NZ9000.

    • Construction of engineering bacteria for conversion of acetophenone to (S)-α-phenylethanol

      2018, 45(12):2576-2584. DOI: 10.13344/j.microbiol.china.180116

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      Abstract:[Background] (S)-α-phenylethanol is a kind of important pharmaceutical intermediate. Conversion of acetophenone into (S)-α-phenylethanol by engineering bacteria has the advantages of strong stereoselectivity and mild transformation conditions. The study of this synthetic method is of great significance for the future green industrial production. [Objective] Construction of engineering bacteria capable of transforming acetophenone into (S)-α-phenylethanol, and the study on its transformation conditions. [Methods] ReADH gene encoding carbonyl reductase and FDH gene encoding formate dehydrogenase were cloned from Rhodococcus erythropolis and Candida boidinii, respectively. The constructed pRSFDuet-ReADH-FDH (R1F2) and pRSFDuet-FDH-ReADH (F1R2) were transformed into Escherichia coli, respectively. The enzymatic assay of ReADH and FDH were analyzed. The optimized transformation conditions to produce (S)-α-phenylethanol were tested. [Results] The enzyme activity of ReADH and FDH in R1F2 were 6.7 U/mL and 7.6 U/mL, respectively. R1F2 had higher catalytic activity than F1R2. The optimum pH of ReADH and FDH were 6.0 and 8.5, respectively. The optimum temperature of the ReADH and FDH were 40 °C and 35 °C, respectively. The optimum pH and temperature for the reaction of acetophenone catalyzed by R1F2 is 7.5 and 30 °C. R1F2 co-expression strain could catalyze the reduction of acetophenone with high substrate concentration, the conversion of 400 mmol/L acetophenone was over 98%, and the e.e. of (S)-α-phenylethanol value was over 99%. [Conclusion] Conversion of acetophenone into (S)-α-phenylethanol by engineered strain has a good prospect for industrial application.

    • Expression, purification and characterization of a novel zearalenone hydrolase from Rhinocladiella mackenziei

      2018, 45(12):2585-2591. DOI: 10.13344/j.microbiol.china.180099

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      Abstract:[Background] Zearalenone (ZEN) is one of the most widely contaminated mycotoxins, which causes huge economic losses to feed industry and animal husbandry. The lactonase ZHD101 is the most widely studied ZEN-detoxifying enzyme. However, its application in industry was limited by the low thermostability. [Objective] In order to realize the application of zearalenone-degrading enzyme in industry, this study explored a ZEN-detoxifying enzyme with more prominent enzymatic properties. [Methods] We found an Rmzhd gene from Rhinocladiella mackenziei CBS 650.93 in the GenBank database, and constructed the pET-46-Rmzhd plasmid. E. coli system was used to express the target protein. The protein was purified by using affinity chromatography and ion exchange purification system. Enzymatic properties were analyzed by high performance liquid chromatography (HPLC). [Results] In this study, we characterized a novel ZEN hydrolase, denoted as RmZHD. RmZHD exhibits the highest activity at pH 8.6 and 45 °C, and has a higher thermostability. Analyzing the structure of RmZHD, we found that the higher contents of salt bridges and solvent-exposed prolines might contribute to higher protein thermostability. [Conclusion] This study provides foundation for improving the industrial applications of zearalenone hydrolases.

    • >Environmental Microbiology
    • Isolation and identification of Bacillus sp. hsn03 with algicidal activity on Microcystis aeruginosa

      2018, 45(12):2592-2602. DOI: 10.13344/j.microbiol.china.180046

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      Abstract:[Background] Cyanobacteria often form water bloom, seriously damage the aquatic environment and threaten aquatic organisms. Microbes can specifically inhibit the growth of cyanobacteria, and be used to control cyanobacteria bloom. [Objective] To isolate and screen bacterial strains with high algicidal ability against harmful algal bloom causing species—— Microcystis aeruginosa 7806, as well as the identification of the physiological, biochemical and molecular characteristics, meanwhile, algicidal mode and activity of the bacterium. [Methods] The physiology and biochemistry characteristics of strain hsn03 were studied by the API test kit. The 16S rRNA gene was sequenced and the phylogenetic tree was constructed. The algicidal activity was investigated by determination of the algicidal effect of bacterial cells, supernatant and bacterial culture on Microcystis aeruginosa 7806. The characteristics of algicidal substance were determined by measuring the algicidal activity of algicidal substance treated by different temperature, pH, protease K, organic solvent extraction, dialysis, as well as FTIR spectroscopy. [Results] Strain hsn03 possessed the highest similarity (99.59%) with the Bacillus sonorensis NBRC101234. Strain hsn03 showed high algicidal activity on M. aeruginosa through secreting extracellular algicidal substances, and the optimum addition amount of bacterial supernatant was 7.0% (v/v). The characteristics of algicidal substance was high thermal stability, pH stability and low molecular weight (500?1 000 Da), which was proved to be not protein and polysaccharide, and algicidal active compound was confirmed to contain triple bond and cumulated double bonds. [Conclusion] The excavation and identification of algicidal bacteria play an important role in enriching the resource of algicidal bacteria to control harmful algal bloom. By studying algicidal methods, effects and characteristics, we will lay the foundation for further application of algicidal bacteria to control Microcystis aeruginosa.

    • Diversity and structure of hypolithic bacteria community of Zhongyang Gobi

      2018, 45(12):2603-2613. DOI: 10.13344/j.microbiol.china.180041

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      Abstract:[Background] Bacteria of hypolithic biological soil crusts (BSCs) in Gobi play an important role in the biogenic elements circling in arid and semi-arid regions. The bacterial community structure and diversity vary greatly due to different climatic and geographical environments. The area of Zhongyang Gobi is large, but little is known so far about the structure and diversity of the hypolithic bacteria community. [Objective] To compared and analyzed the similarities and differences of bacterial community structure and diversity in hypolithic BSCs and bare soil around in the Zhongyang Gobi, and discover the effect of environmental factors on bacterial community and the interaction relationships among the bacteria. [Methods] Illumina MiSeq was applied to sequence 16S rRNA gene, bioinformatics approach was used to reveal the diversity and structure of the bacteria community, the co-occurrence network was constructed based on CoNet software and Cytoscape 3.5.1 to visualize the network. [Results] The contents of available phosphorus (AP), available nitrogen (AN) and chlorophyll a (Chl a) of the soil were significantly higher (the most of 471% higher), while pH were slightly lower in the BSCs than that in the bare soil. The bacteria was mostly dominated by the phylum of Cyanobacteria (45.85%?53.77%) and by the genera Trichocoleus, Chroococcidiopsis and unknown one belonged to Class Cyanobacteria (9.25%?18.42%) in the hypolithic BSCs, and did by the phylum of Actinobacteria (38.82%?44.69%) and by the genera Arthobacter, Rubrobacter and three unknown genera belonged to the phylum of Actinobacteria or Acidobacteria (>5%) in the adjacent bare soil. Among all the soil physicochemical factors, AP had the most significantey influence on the composition of hypolithic bacterial communities. Except the genera of Actognophytocola and one unclassified of Class Cyanobacteria, there were strong interaction existed among other phylotypes, in which the co-occurrence relationship was dominant (about 60%) and the nodes were all belonged to Phylum Cyanobacteria and Class α-Proteobacteria with higher degree, closeness and betweenness centralities. [Conclusion] Bacterial diversity and community structure of hypolithic BSCs is remarkably distinguished from that of bare soil in the Zhongyang Gobi. Members of Phylum Cyanobacteria and Class α-Proteobacteria are the primary and most important driver in the early development of hypolithic BSCs in the Zhongyang Gobi.

    • Identification of algicidal bacterium Sp37

      2018, 45(12):2614-2623. DOI: 10.13344/j.microbiol.china.180028

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      Abstract:[Background] Phytoplankton blooms are a worldwide problem that brings a serious threat to environment and human health. It is urgent to control bloom through effective methods. Algicidal bacteria have been considered as an effective strategy to control bloom because they can kill algae. [Objective] To isolate and identify Microcystis aeruginosa and its algicidal bacteria from Dianchi Lake, and further analyze the algicidal effect and inhibition mechanism of algicidal bacteria against M. aeruginosa. [Methods] Bacteria were isolated by dilution separation method and identified based on 16S rRNA genes. Algae were isolated by dilution separation and capillary separation methods, and identified based on cpcBA and 18S rRNA genes. Algicidal efficiency was calculated based on the chlorophyll-a concentration of algal culture, which was measured with heat-ethanol extraction method. Determination of CAT (catalase), GSH (glutathione) and MDA (malondialdehyde) were used to investigate the algal response of antioxidant system to the algicidal bacteria. [Results] In total 11 Microcystis and 17 algicidal bacteria strains were isolated, M. aeruginosa DCM4 and Bacillus siamensis Sp37 were selected for the further research. Sp37 showed a strong algicidal activity against M. aeruginosa DCM4 (Ae=92.4%±1.5%, t=4 d), and it also can kill M. flos-aquae and M. wesenbergii of Cyanophyta, but not Monoraphidium and Scenedesmus of Chlorophyta; there is no significant difference of algicidal ratio (P>0.05) between the culture and cell-free filtrate of Sp37 (86.8%±4.3% and 81.1%±2.2% (t=4 d), respectively), while that of washed cells were much lower (25.4%±7.3%); Sp37 cell-free filtrates that treated at different temperature and pH conditions displayed no obvious difference of algicidal effects; Cell-free filtrate of Sp37 caused great changes of CAT, GSH and MDA content of DCM4. [Conclusion] Sp37 had strong algicidal effect on M. aeruginosa DCM4, and it is likely can kill Microcystis selectively. Sp37 kills M. aeruginosa DCM4 by releasing algicidal substance(s) which has (have) good thermal and acid-base stability. Cell-free filtrate of Sp37 can activate antioxidant systems and destroy cell membrane of DCM4, and ultimately cause algal cell death.

    • Effects of nitrogen sources and levels on the growth and accumulation of lipids and arachidonic acid of three coccoid green algae

      2018, 45(12):2624-2638. DOI: 10.13344/j.microbiol.china.180080

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      Abstract:[Background] Lobosphaera incisa Reisigl is a unicellular coccoid green algae, one of the richest plant source of arachidonic acid (AA). However, its taxonomic position has been unclear so far. [Objective] The taxonomy of three coccoid green algae (SAG2468, SAG2043 and H4301) with similar morphology were clarified, and the effects of different nitrogen sources (NaNO3, (NH2)2CO, NH4HCO3, (NH4)2CO3, NH4NO3, (NH4)2SO4 and NH4Cl) and levels (18 mmol/L, 3 mmol/L) on their lipids and AA accumulation were analysed. [Methods] Molecular and morphology identification were used to analyse the taxonomic position of the three strains; dry weight, gravimeter and gas chromatographic analyses were used to determine the biomass concentration and the content of total lipids (TLs) and AA. [Results] The three strains all belonged to the genus of Lobosphaera. Parietochloris incisa SAG2468 was revised to Lobosphaera incisa; SAG2468 and H4301 were the different geographical strains of L. incisa. Myrmecia bisecta SAG2043 was revised to Lobosphaera bisecta. The high (18 mmol/L) and low (3 mmol/L) nitrogen concentration of NaNO3 and (NH2)2CO were all suitable for the growth of these three strains. Ammonium as nitrogen source inhibited the growth of algae, and the higher the concentration, the more inhibition. Low-nitrogen concentration could promote TLs and AA accumulation in all strains significantly (P<0.05). The remarkable TLs and AA volumetric productivities of 142.15 mg/(L·d) and 35.51 mg/(L·d) were obtained in SAG2043 at 3 mmol/L NaNO3, significantly higher than those of H4301 and SAG2468 (P<0.05). Under these conditions, the corresponding biomass concentration was 4.9 g/L, the TLs reached 43.49% and the AA contents were up to 10.86% of dry weight and 31.75% of the total fatty acids in SAG2043. [Conclusion] SAG2043 showed a more prominent advantage to AA production.

    • Enhancing cell growth and lipid production of Chlamydomonas reinhardtii by co-culturing with the fungus Simplicillium lanosoniveum

      2018, 45(12):2639-2647. DOI: 10.13344/j.microbiol.china.180096

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      Abstract:[Background] Algae are the major feed-stocks for biofuel production, and some fungi and bacteria can establish symbiotic relationships with algae and promote their productivities. Hence, co-culture of algae with fungi or bacteria becomes the research hotspot worldwide. [Objective] To study the effects of cyanobacterium-symbiotic fungus Simplicillium lanosoniveum on the cell growth and lipid synthesis of Chlamydomonas reinhardtii. [Methods] C. reinhardtii was co-cultured with Simplicillium lanosoniveum. [Results] Compared to C. reinhardtii monoculture, the specific growth rate (0.20 d?1), cell productivity (0.17 g/(L·d)) and biomass (2.85 g/L) of C. reinhardtii in co-culture increased 10.3%, 51.3% and 55.7% respectively. The specific lipid synthetic rate (0.68 mg/(g·d)), lipid productivity (1.95 mg/(L·d)) and lipid content (220.4 mg/g) increased 33.3%, 107.5% and 32.0% respectively. Also, the proportions of saturated fatty acids and monounsaturated fatty acids C18-1 and C18-2 in the lipid elevated, indicating the suitability for biofuel production. [Conclusion] Simplicillium lanosoniveum promotes cell growth and lipid synthesis of C. reinhardtii, and thus algae-fungi co-cultivation can be considered in biofuel feedstock production.

    • Consistency analysis of MiSeq sequencing data of 16S rRNA genes from different biotech companies

      2018, 45(12):2648-2661. DOI: 10.13344/j.microbiol.china.171087

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      Abstract:[Background] High-throughput sequencing technology has been widely used in the research field of environmental microbiology. Due to sequencing platform based on different principles, and personalized service biotech companies provided, huge amounts of various sequencing data are emerged. Although personalized service is good to meet customers’ different requirements, there is a widespread concern if the sequencing data from different sequencing platforms or different companies could be equally treated. [Objective] The aim of this study is to explore the impacts of different sequencing conditions and sequencing depths on the final sequencing data of the same sample using MiSeq sequencing platform, and further to find out the reasons for the differences, and the subsequent effects of these differences on the experimental data. [Methods] Sediment samples were collected from Songmenshan Region, Nanjishan, Raohe River and Baishazhou of the Poyang Lake. High-throughput sequencing of 16S rRNA gene V3?V4 region was performed in two biotech companies with different sequencing depths, and two sets of data were compared. [Results] Two sets of data showed highly similarity in microbial community structure, but the abundance difference between the rare species resulted in a different pattern in the PCoA and cluster analysis. Co-occurrence network revealed that the data with higher sequencing depth could reflect more complex interactions between and within microbial taxa. Some rare species such as Deferribacteres and Lentisphaerae were found to be important for the community eco-function. A total of 14 categories of differentiated metabolism were found between two datasets by METAGEN assist functional forecast method, including Atrazine metabolism, Chitin degradation, Sulfate reducer, Nitrogen fixation and so on. [Conclusion] The impacts of different sequencing environments on experimental data can be reduced or even eliminated by data quality control processes, but different sequencing depths have a significant impact on the sequencing data. Increasing the sequencing depth can significantly improve the richness of rare species, and thus supply a comprehensive knowledge of microbial community function.

    • >Fundamentals of Microbiology
    • Symbiotic Escherichia coli promotes the developmental timing of Drosophila melanogaster

      2018, 45(12):2662-2672. DOI: 10.13344/j.microbiol.china.180006

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      Abstract:[Background] The symbiotic microbiota profoundly affects many aspects of host physiology, but the diversity and complexity of microbial community make it difficult to explore the underlying mechanism in vertebrates. Fruit fly Drosophila provides us a germ-free and gnotobiotic model to investigate the interaction of microbes and hosts. [Objective] To isolate and identify Escherichia coli from Drosophila melanogaster gut and investigate the effects of E. coli on the development of hosts. [Methods] E. coli was isolated with selective medium and identified with BLASTn analysis of 16S rRNA gene. In vitro and in vivo co-existence test were used to verify the symbiosis. Through the developmental timing and growth rate experiments, the effect of E. coli on hosts’ development were investigated. Real-time quantitative PCR were used to assess gene expression levels of PTTH and insulin signaling pathways. [Results] We isolated and identified indigenous strains of E. coli in the guts of both lab-reared and wild-captured Drosophila. E. coli was co-cultured with commensal Lactobacillus plantarum in vitro, and in vivo colonized the fly gut, indicating that E. coli was one symbiotic member of the bacterial community of Drosophila. Moreover, E. coli facilitated the development of Drosophila by accelerating the growth rate. At the molecular level, E. coli significantly stimulated the activity of PTTH and insulin signaling that is essential for the larval/pupal transmission in Drosophila. [Conclusion] E. coli was symbiotic bacteria of Drosophila and promoted the development of Drosophila.

    • Diversity of endophytic actinobacteria isolated from medicinal plants collected from Guizhou Province

      2018, 45(12):2673-2683. DOI: 10.13344/j.microbiol.china.180074

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      Abstract:[Background] Endophyte from medicinal plants were important resource for discovery of new antibiotics due to their ability of producing a wide variety of secondary metabolites with good activity. Concerning the endophytic fungi, many studies have been carried out, but few reported about endophytic actinobacteria. [Objective] This study is aimed at analyzing the diversity of actinobacteria isolated from medicinal plants in Guizhou Province. [Methods] Various tissues from 8 kinds of medicinal plants as samples were collected in Guizhou Province, China. Samples were grinded into powders after surface sterilization and cultured on 9 different media for strain isolation. To determine the phylogenetic position of the strains, 16S rRNA gene sequences were obtained by PCR amplification and direct sequencing, then compared with EzBioCloud database, finally carried out phylogenetic analysis based on 16S rRNA gene sequences. [Results] A total of 62 strains were obtained, 57 of which were actinobacteria and were affiliated to 16 genera 8 families 5 order, contained genus Curtobacterium, Streptomyces, Microbacterium, Cellulomonas, Plantibacter, Promicromonospora, Kineococcus, Agrococcus, Patulibacter, Aeromicrobium, Labedella, Frondihabitans, Frigoribacterium, Arthrobacter, Kineosporia and Amnibacterium. The dominant genus is Curtobacterium. The highest similarity of 16S rRNA gene sequence between strain M8JJ-5 and the validly described species Amnibacterium kyonggiense KSL51201-037T (FJ527819), between strain M2KJ-4 and the validly described species Aeromicrobium fastidiosum DSM10552T (Z78209) was 97.29% and 98.95%, respectively, which indicated that the strains M8JJ-5 and M2KJ-4 were potential new taxa. [Conclusion] The study demonstrates that diversity of endophytic actinobacteria from medicinal plants are rich and medicinal plants as a special resource for discovery of new pharmaceutical microorganisms deserved to be further explored.

    • >Agricultural Microbiology
    • Screening, identification and preliminary study on biocontrol effect of antagonistic Bacillus against the pathogen of Valsa mali for apple tree

      2018, 45(12):2684-2694. DOI: 10.13344/j.microbiol.china.180038

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      Abstract:[Background] Apple tree canker is widespread and dangerous. Currently, chemical control is the main way to prevent and cure it. However, there are some drawbacks. Therefore, it is urgent to find an effective and safe prevention and control measure. [Objective] In order to select Bacillus isolated in Saline-alkali soil from Dunhuang that showed significantly antagonistic effect on the Valsa mali growth. [Methods] The antifungal activities of selected Bacillus were investigated by dual culture assar and colony diameter assay, respectively. In addition, the control effect of antagonistic bacteria strains against Valsa mali in detached apple twigs in vitro and selection for medium were determined and the antagonistic Bacillus was identified based on morphological, physiological and biochemical characteristics and 16S rRNA gene phylogenetic analysis. [Results] The results revealed that strain 7-2-1-2 exhibited a strong antagonistic activity against V. mali, inhibiting significantly the germination of conidia and the growth of hyphae and especially the inhibitory rate of strain 7-2-1-2 was 86.18% and 73.2% in second screening. Strain 7-2-1-2 fermentation broth applied prior to wound inoculation of apple twigs with V. mali resulted in total inhibition of infection. The optimum growth medium of strain 7-2-1-2 was NBY. Strain 7-2-1-2 was identified as Bacillus subtilis subsp. spizizenii. [Conclusion] These results indicated that the strain 7-2-1-2 promised to provide a new biological control method in containing apple canker.

    • >Food Microbiology
    • Comparison of the virulence genes of Enterococcus faecalis isolated from different environments

      2018, 45(12):2695-2707. DOI: 10.13344/j.microbiol.china.180026

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      Abstract:[Background] Enterococcus faecalis, as an important lactic acid bacteria, is widely used in dairy products and pharmacy. However, many E. faecalis isolates are opportunistic pathogen. So it is important to analyze of virulence genes in E. faecalis. [Objective] To study the virulence genes of E. faecalis from different environments and evaluate the safety of E. faecalis isolated from naturally fermented dairy products. [Methods] Comparative genomic analysis was used to determine the virulence genes of 107 strains isolated from dairy products, blood, urine, feces and water. Principal component analysis (PCA) was used to compare the virulence genes of different isolates. The habitat-specific virulence genes were screened by Pearson’s chi-squared test. [Results] A total of 88 virulence genes were identified in 107 genomes of E. faecalis isolates. These virulence genes were mainly involved in adhesion. There was no significant difference in the number of virulence genes between dairy isolates and the other isolates. [Conclusion] The results indicated that the E. faecalis isolated from dairy products were also at risk of pathogenicity. The safety of E. faecalis must be evaluated before using in the food industry.

    • Biodiversity of wine-related yeasts isolated from Shangri-La wine-producing region of Yunnan

      2018, 45(12):2708-2721. DOI: 10.13344/j.microbiol.china.180065

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      Abstract:[Background] Shangri-La wine-producing region of Yunnan is located in the Three Parallel Rivers National Park, and its microbial resources are abundant. The variety of wild-yeasts associated with wine making is also very diverse. [Objective] To study the diversity of yeasts and the genetic diversity of Saccharomyces cerevisiae in Shangri-La wine-producing region. [Methods] Five vineyards from the Shangri-La Jinsha River and Lancang River were selected to collect ripe grape samples and separate the yeasts from the grape peel and fermentation process. The yeast species were identified by WL (Wallerstein laboratory nutrient agar) identification medium and 26S rDNA D1/D2 domain sequence analysis. The genetic diversity of S. cerevisiae was studied by SSR molecular markers. [Results] a total of 230 wild yeasts isolated from grape berries were identified as 13 genera and 18 species, of which 10 species were found for the first time in Shangri-La. Genetic diversity of 47 S. cerevisiae strains was analyzed by SSR molecular markers, and 47 strains of S. cerevisiae were divided into 24 genotypes. 70 alleles were observed by microsatellite markers among the 47 strains. The average polymorphism information content (PIC) was 0.640. The mean observed heterozygosity (Ho) was 0.166, and the mean expected heterozygosity (He) was 0.693. [Conclusion] There are abundant yeast resources in the Shangri-La Wine-producing region, which shows the high species diversity of yeast and moderate genetic diversity of S. cerevisiae. Study on the diversity of yeast in this area, it aims to lay a foundation for the conservation and utilization of the diversity of Shangri-La’s yeast resources.

    • Screening of lactic acid bacteria with good characteristics in natural fermented sowthistle from Ordos

      2018, 45(12):2722-2730. DOI: 10.13344/j.microbiol.china.180101

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      Abstract:[Background] The flavor and effect of traditional natural fermented sowthistle in Ordos are unique, but little is known about the species and biological study on lactic acid bacteria in fermented sowthistle. [Objective] In order to obtain lactic acid bacteria used in industrial production. [Methods] Through the determination of the total acidity, the high-yield acid strains were screened. The morphology, physiological and biochemical characteristics, and 16S rRNA genes were studied. [Results] All of these five strains are Lactobacillus plantarum, and the temperature range of growth is wide, and they had the characteristics of acid resistance, alkali resistance, salt tolerance and heat resistance. [Conclusion] The experimental strains have good physiological characteristics to be used in the food industry.

    • >REVIEWS
    • Sclerotia of plant pathogenic fungi

      2018, 45(12):2762-2768. DOI: 10.13344/j.microbiol.china.180117

      Abstract (1471) HTML (4224) PDF 1.77 M (2342) Comment (0) Favorites

      Abstract:Fungal sclerotium is a dormant structure formed by the branching, interweaving and aggregation of hyphae under the adverse environments, which plays crucially biological and ecological roles in the fungal life cycle and disease cycle. Many devastating plant pathogenic fungi form sclerotia, by which the fungi survive through the adverse environments. Sclerotia germinate and produce apothecia and spores or hyphae that subsequently cause plant infection. Herein, species diversity, biological characterization, melanization, and molecular regulation of sclerotium-forming fungi were comprehensively summarized to provide novel potentials of fungal sclerotia in mycology, plant pathology, and medicinal mycology.

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