• Volume 43,Issue 8,2016 Table of Contents
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    • >NEWS AND VIEWS
    • A new tool for the genome-editing in E. coli

      2016, 43(8):1863-1863. DOI: 10.13344/j.microbiol.china.168008

      Abstract (1248) HTML (635) PDF 235.34 K (2342) Comment (0) Favorites

      Abstract:

    • >On Focus
    • Knocking out phosphoenolpyruvate carboxylase gene by CRISPR/Cas and its influence on fatty acid metabolism in Escherichia coli

      2016, 43(8):1864-1871. DOI: 10.13344/j.microbiol.china.150732

      Abstract (1737) HTML (908) PDF 923.90 K (3407) Comment (0) Favorites

      Abstract:[Objective] To obtain recombinant strain of Escherichia coli to study fatty acid metabolism, the newly developed CRISPR/Cas system was used to modify the genome of E. coli MG1655. [Methods] A binary vector was constructed with CRISPR/Cas and λ-Red recombinase. Donor genes were then knockout through overlapping PCR. The recombinant vector plasmid was subsequently electro-transformed into E. coli MG1655 to edit the gene associated with fatty acid metabolism. Furthermore, fatty acid contents were measured quantitatively by gas chromatography-mass spectrometry (GC-MS). [Results] Six recombinant E. coli strains were obtained, including ΔPEPC (partly knock-out), ΔPEPC, ΔPEPC(partly knock-out)-ΔFadD, ΔPEPC-ΔFadD and ΔFadD. Compared to the wild one, all reconstructed strains showed higher yields of total fatty acids with maximum increase of 3.7%, whereas the yield of mutant strain ΔPEPC was lower than expected. The fatty acid of 11:0, 12:0, 13:0, 14:0, 15:0, 16:0, 17:1, 17:0, 18:0 were detected in all strains, and 16:0, 17:0, 18:0 were dominant. [Conclusion] The CRISPR/Cas system with Lambda-Red recombinases was an efficient and rapid tool to modify genes of E. coli, and opened a new way to construct recombinant E. coli strains.

    • >Commentary
    • Analysis of resistant bacteria —“Only things of a national touch can prevail in the world”

      2016, 43(8):1872-1872. DOI: 10.13344/j.microbiol.china.169008

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      Abstract:

    • >Industrial Microbiology
    • Selection of endogenous expression elements from Corynebactrium glutamicum

      2016, 43(8):1671-1678. DOI: 10.13344/j.microbiol.china.160169

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      Abstract:[Objective] We constructed a Corynebacterium glutamicum expression-element probe vector and selected sequences that can activate expression of protein. [Methods] Based on the classic plasmid pXMJ19, we set the inserted position with the new method of Golden Gate cloning strategy that can connect material sequence to the report gene seamlessly, avoiding remaining unwanted sequence that may interfere the measurement of expression elements effect. By analyzing the transcriptomic data of C. glutamicum BZH001 cultivated under different dissolved oxygen level in our previous study, we screened six genes stabilizing in high transcriptional level, analyzed their promoter regions and 5′UTR regions by the promoter software prediction, and then amplified the promoter sequences associated with 5′UTR regions of the six genes. The activities of these expression elements were measured using the reporter gene egfp in the probe vector. [Results] Five expression elements with different performances were achieved, and the best one could reach over 3 500 RFU/OD600 of fluorescent intensity. [Conclusion] Associated with the transcriptomic data, we can screen effective expression elements using this probe vector, which can provide more genetic parts for the future genetic engineering and construction of biological system in Corynebacterium glutamicum.

    • >Environmental Microbiology
    • Identification and characterization of a denitrifying bacterium Pseudomonas fluorescens

      2016, 43(8):1679-1689. DOI: 10.13344/j.microbiol.china.150717

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      Abstract:[Objective] In order to isolate a bacterium, with high efficiency denitrifying, from activated sludge samples. [Methods] The strain, was named L2, isolated through domestication enrichment, purification, preliminary and secondary screening, which was identified based on its morphology characteristics, and 16S rRNA gene sequence analysis. we systematic studied its various denitrification characteristics as follows: inoculation volume, temperature, pH, salt concentration, carbon source, dissolved oxygen (DO), carbon nitrogen ratio (C/N) and initial nitrate concentration. [Results] The strain was identified as Pseudomonas fluorescens, and the results showed that the most suitable denitrification conditions of the strain are inoculation volume of 10%, temperature of 20 °C, with the poor thermal stability, pH of 7.0, salt concentration of 0.5%, carbon source of glucose, C/N of 5.0, and it can tolerate high initial nitrate concentration. [Conclusion] This Pseudomonas fluorescens, which had good resistance to low temperature, high tolerance to initial nitrate concentration, tolerance low carbon nitrogen ratio, was a facultative anaerobic efficient denitrifying bacterium.

    • >Microbial Genetics
    • Cloning and functional analysis of 5 glyphosate-response-genes in mutant Enterobacter NRS-1 strain

      2016, 43(8):1690-1698. DOI: 10.13344/j.microbiol.china.150675

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      Abstract:[Objective] The mechanism of microbe’s tolerance to glyphosate stress is controlled by complex genetic systems including the target gene and many related regulation genes. In the present study, five different expression genes (DEGs) of a mutant Enterobacter strain NRS-1 were identified, the function and interaction were investigated to reveal the role of non-target genes on glyphosate resistance. The work intends to provide potential gene resources for transgenic breeding of glyphosate resistance. [Methods] DEGs of NRS-1 may play a role on the processes of protein biosynthesis, metabolism, cell membrane etc. Accordingly, five DEGs, encoding elongation factor G (fusA), periplasmic protein (osmY), ornithine carbamoyltransferase 2, chain F (argF), thymidine phosphorylase (deoA), succinate dehydrogenase flavoprotein subunit (sdhA), were selected. The sequences of the genes were obtained through T-A cloning, the functions were detected though analyses of prokaryotic expression and transgenic programs. Moreover, the interaction between five genes and the glyphosate-target gene aroA (5-enolpyruvylshikimate-3-phosphate synthase) was also detected using bacteria two-hybrid system and KEGG pathway bioinformatic analysis. [Results] The characteristic of five genes was revealed. According to the performance of prokaryotic expression and transgenic Arabidopsis thaliana, genes were confirmed to help cell to improve the tolerance to glyphosate. The genes argF, deoA have better resistance trait than other genes, similar as the target gene aroA. It seems that the pathways including the synthesis and metabolism of aromatic amino acids, amine acids containing thymine and arginine might be important for NRS-1 to tolerant to glyphosate. Regulation network showed a complicated relationship between the five selected gene and aroA, and no direct interaction was found between them. [Conclusion] The selected five genes had positive effects on the resistance to glyphosate stress, among them, argF, deoA were better than others. Complex gene interaction could be found between genes and aroA.

    • >Agricultural Microbiology
    • Isolation, identification and anti-oxidative activity of metabolites from oxygen-tolerant mutant strain Aeroto-AUH-JLC140 capable of biotransforming daidzein

      2016, 43(8):1699-1707. DOI: 10.13344/j.microbiol.china.150609

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      Abstract:[Objective] Compared to the original bacterium strain AUH-JLC140, its oxygen-tolerant mutant strain named Aeroto-AUH-JLC140 was able to produce an unknown substance. In addition, the production of the unknown substance was not associated with the addition of the substrate daidzein to the cultural medium. The objective of this study was to isolate and identify the unknown substance, and then to study the kinetics and the anti-oxidative capacity of the unknown substance produced by the oxygen-tolerant mutant strain Aeroto-AUH-JLC140. [Methods] The unknown substance was isolated by high-performance liquid chromatography (HPLC) method. Based on the analyses of the UV spectrum, electrospray ionization mass spectrometry (ESI-MS) as well as the 1H and 13C nuclear magnetic resonance spectroscopy, the unknown substance was identified. The anti-oxidative activity of the unknown substance was determined by the 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) scavenging activity. [Results] The unknown substance produced by the oxygen-tolerant mutant strain Aeroto-AUH-JLC140 was identified as indole. Kinetics study showed that indole reached to the highest after being incubated for 15 h and the highest amount of indole in the cultural broth was 19.89 mg/L. Study on free radical-scavenging activity indicated that 0.2 mmol/L (i.e. 23.40 mg/L) of indole not only showed an obvious DPPH radical-scavenging activity but also decreased the oxidation-reduction potential (ORP) of brain heart infusion (BHI) liquid medium. [Conclusion] The unknown metabolite produced by the oxygen-tolerant mutant strain Aeroto-AUH-JLC140 is indole. The amount of indole produced by strain Aeroto-AUH-JLC140 can decrease the ORP of BHI liquid medium, which subsequently provides a suitable microenvironment with lower ORP for growth of strain Aeroto-AUH-JLC140 in the presence of atmospheric oxygen.

    • Field assessment of symbiotic efficiency, growth-promoting ability and phylogeny of soybean rhizobial strain SCAUs8

      2016, 43(8):1708-1714. DOI: 10.13344/j.microbiol.china.150613

      Abstract (1469) HTML (844) PDF 479.41 K (2149) Comment (0) Favorites

      Abstract:[Objective] Soybean is the important legume crops in China. We can reduce chemical nitrogen fertilizer effectively by soybean-rhizobia symbiosis. The former screened soybean rhizobial strain SCAUs8 was used in field inoculation to two important soybean planting regions, in Sichuan. In addition, we identified the taxonomic position of this strain. [Methods] Field experiments were conducted to investigate the effect on soybean production by inoculating soybean rhizobia strain SCAUs8 in Sichuan hilly area and Panxi area. We studied the stress-tolerance of SCAUs8 with the point inoculation method. Growth-promoting ability was determined through Salkowski colorimetric method. Multilocus sequence analyses (16S rRNA, atpD, recA, glnⅡ, nodC, nifH) were amplied to identify the phylogenetic position of SCAUs8. [Results] Field experiments showed that plant fresh weight and dry weight inoculated with SCAUs8 were significantly higher than that of no inoculation control (CK). The yield increased significantly by 21.4%?29.7% compared with CK. The strain SCAUs8 could grow at pH 5.0?10.0. It could be resistant to 0.5% NaCl, and the growth temperature range was 10?40 °C. Growth-promoting capability of secreting heteroauxinc (IAA), was also be detected. By analyses of 16S rRNA, atpD, recA, glnⅡ, nodC and nifH sequences, we found that SCAUs8 clustered closely to Bradyrhizobium diazoefficiens USDA110T with 100% similarities. [Conclusion] SCAUs8 was a good candidate matching with main planting cultivars of Sichuan soybean. This strain had alkaline and acid tolerance, wide growth temperature range and could secrete growth-promoting IAA, although the salt tolerance of it was weak. Phylogenic study proved that strain SCAUs8 was assigned as B. diazoeffciens.

    • Isolation, identification and characterization of an antagonistic bacterium against Penicillium expansum

      2016, 43(8):1715-1724. DOI: 10.13344/j.microbiol.china.150662

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      Abstract:[Objective] In order to obtain an antagonistic strain which can effectively inhibit the growth of Penicillium expansum and to identify the antifungal substances produced by this strain. [Methods] The antagonistic strain BA-16 that could inhibit Penicillium expansum growth was isolated from apple surface and then identified through phenotypic, physiological, biochemical and phylogenetic (16S rRNA gene) analyses. Three primers were designed on the basis of the relevant genes and PCR was done. After the PCR product was purified and sequenced, the obtained sequences were analyzed by BLAST. The crude anti-fungal extracts were prepared by using acidic precipitation method and the active antagonistic substances were purified and identified by HPLC and MALDI-TOF-MS analysis. [Results] The strain BA-16 was identified as Bacillus amyloliquefaciens. PCR detection and BLAST analysis revealed that strain BA-16 expressed the sfp and fenB genes. The HPLC and MS analysis results demonstrated that the fermentation broth of the strain contained fengycin and surfactin. Moreover, fengycin played an important role in inhibiting Penicillium expansum. [Conclusion] The strain BA-16 will be a potential biological control agent against blue mold decay in apples.

    • >Food Microbiology
    • Study on the antibiotic resistance genes diversity of fermented products in Inner Mongolia Xilin Gol pasturing area

      2016, 43(8):1725-1731. DOI: 10.13344/j.microbiol.china.150639

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      Abstract:[Objective] This study was aimed at revealing the usage condition of antibiotics at Xilin Gol pasturing area through researching on the diversity of antibiotic resistance genes (ARGs) in fermented dairy products. Furthermore, it will accumulate basic data for interpretating the mechanism of source, transmission and diffusion of antibiotics. [Methods] Quantitative polymerase chain reaction (q-PCR) was applied to quantify the abundance of resistance genes in fermented dairy product (6 of yoghurt and 5 of koumiss) collected in Xilin Gol pasturing area. [Results] Twenty-two ARGs were detected in nearly all samples,ranging from (1.028±0.338) to (8.648±0.087) lg(copies/mL). By comparing the yoghurt and koumiss samples, the abundance of ermB, strA, strB, gyrA, tetO, yidy, cat genes in yoghurt were significantly higher than that of the latter (P<0.05). No significant differences were found in the abundance of the rest of fifteen ARGs between two groups of samples. [Conclusion] The study demonstrated that the fermented dairy products served as a reservoir of ARGs. It is necessary for us to study the antibiotic resistance of fermented dairy products from the phenotypic and genetic level.

    • Effect of disrupting ILV2 gene on growth and diacetyl metabolism of brewer’s yeast

      2016, 43(8):1732-1738. DOI: 10.13344/j.microbiol.china.150676

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      Abstract:[Objective] The purpose is to reduce diacetyl content in beer fermentation liquor by molecular biology methods and improve sensory quality of beer. [Methods] Based on S2 (Saccharomyces cerevisiae, tetraploid), mutant strains, including single ILV2 allele disruption (QI2-1) and two ILV2 alleles disruption (QI2-2) were constructed by homologous recombination to destroy acetyl lactic acid synthase gene (ILV2), and beer fermentation experiment was carried on. [Results] It could reduce?the initial growth rate of strains because of the disruption of ILV2 gene, especially QI2-2, while the growth rate of mutant strains was same as the host strain after 12 h. Beer fermentation experiment showed that ILV2 gene could reduce the production of diacetyl, and the diacetyl peak and diacetyl concentration decreased by 17.50% and 17.83% in QI2-1, 51.67% and 45.65% in QI2-2, respectively, compared with that in the host strain S2. Other indicators such as alcohol, beer fermentation degree, residual sugar and flavor changed slightly, but they were in the range of high quality beer suggested, and conformed to the requirements of the index of beer fermentation. [Conclusion] It is an effective method to reduce diacetyl content and improve the quality of beer by constructing low-diacetyl strains using homologous recombination with the disruption of Part of ILV2 gene, and it has certain actual application value.

    • >Veterinary Microbiology
    • Microbial diversity in the gastrointestinal tract of Hipposideros pratti

      2016, 43(8):1739-1745. DOI: 10.13344/j.microbiol.china.150654

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      Abstract:[Objective] We studied the bacterial diversity and pathogenicity in the gastrointestinal tract of Hipposideros pratti. [Methods] The 16S rRNA gene V1?V2 genes were sequenced by using MiSeq high-throughput sequencing to study the bacterial community in stomach and intestine of H. pratti. MG-RAST V3.3.6 was used to evaluate the abundance, diversity and operational taxonomic units (OTUs) of the bacteria. [Results] In total 144 998 and 275 274 original sequences, and 48 332 and 91 758 high quality reads were obtained from the stomach and intestine of H. pratti. They belonged to 894 and 756 OTUs. The bacterial abundance indices (Chao, 1 490; ACE, 2 221) and Shannon diversity index (2.405) in the stomach were lower than those of intestinal tract (Chao, 2 051; ACE, 3 556; Shannon, 2.407). On the contrary, Simpson diversity index (0.168) in the stomach was lower than that of intestinal tract (0.151). Phylogenetic analysis results show that the gastrointestinal bacteria were mainly distributed in 6 Phyla, dominated by Proteobacteria (56.4% in stomach and 46.0% in intestines) and Firmicutes (40.7% in stomach and 49.2% in intestines). There were 24 genera above 0.1% in stomach and intestine of H. pratti. Lactococcus and Hafnia were the 2 dominant bacterial genera in stomach accounting for 26.1% and 21.0% respectively. Enterococcus and Salmonella were 2 dominant genera in intestine accounting for 15.2% and 12.7%, respectively. Unfortunately, these dominant bacteria were human pathogenic bacteria or opportunistic pathogen. [Conclusion] H. pratti carried a large number of human pathogens and thus should be paid more attentions to prevent transmission of diseases from the bats to human.

    • Construction of sub-genomic replicon of foot-and-mouth disease containing EGFP reporter gene

      2016, 43(8):1746-1752. DOI: 10.13344/j.microbiol.china.150673

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      Abstract:[Objective] To construct sub-genomic replicon system of foot-and-mouth disease containing EGFP reporter gene. [Methods] Based on the infectious clone of serotype O FMDV, the subgenomic replicon FMDV-EGFP was constructed by replacing Lb and P1 gene of FMDV with EGFP reporter gene using fusion PCR method. The stability of replicon was detected by a series of transformation and sequencing. The replicon linearized with Not I was transfected into BSR/T7 cells expressing T7 RNA polymerase using liposome mediation. EGFP expression of different time of transfected cells was examined using fluorescence microscopy, the ability of self-replication and expression of protein of replicon were detected by flow analysis, immunofluorescence assay, RT-PCR and Western blot. [Results] The replicon vector was stable by a serious of transformation and sequencing. After 3 h transfection, the EGFP fluorescence could be obviously observed under the fluorescence microscope, the level of EGFP protein was increased gradually as transfection time went on. The result of flow cytometry analysis showed that 6.0% transfected cells were able to generate fluorescence, indicating the effective expression of EGFP protein of replicon vector. In addition, the results of immunofluorescence, RT-PCR and Western blot assay demonstrated that the replicon could autonomously replicate and express non-structural protein of FMDV. [Conclusion] The construction of sub-genomic replicon of FMDV expressing EGFP reporter gene provides a foundation to study viral replication and translation mechanism and antiviral drugs.

    • >Pharmaceutical Microbiology
    • Diversity and antimicrobial activity of endophytic actinobacteria isolated from eumangroves collected in Dongzhaigang of Hainan Province

      2016, 43(8):1753-1765. DOI: 10.13344/j.microbiol.china.150680

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      Abstract:[Objective] To study the diversity and bioactivity of actinobacteria isolated from Mangroves in Dongzhaigang, Hainan. [Methods] Various tissues from 14 kinds of mangrove plants as samples were collected from Dongzhaigang National Nature Reserve in Hainan, China. Samples were grinded into powders after surface sterilization and cultured on 10 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 gene database. The antifungal and antibacterial activities of the crude extracts were tested by disk diffusion method. Screening of PKS I, PKS II and NRPS genes was carried out by PCR and agarose electrophoresis. [Results] 146 strains affiliated to 18 genera were obtained. The dominant genus is Streptomyces. The highest similarity of 16S rRNA gene sequence between strain S3Cf-2 and the validly described species Couchioplanes caeruleus DSM44103T, between strain S3Af-1 and the validly described species Microlunatus terrae BS6T was 97.45% and 97.43%, respectively, which indicated that the strains S3Cf-2 and S3Af-1 were potential new taxa. The antimicrobial activity of crude extracts from the fermentation broths were tested against different pathogenic microorganisms by paper-disc diffusion method. Antimicrobial test showed that 40 out of 46 strains exhibited positive results in at least one antifungal or antibacterial assay, and the total positive rate was 86.96%. Thirty eight in 40 strains with antimicrobial activity, probably, had at least one antibiotic biosynthetic gene including NRPS, PKS I and PKS II, and 14 strains have all the three kinds of biosynthetic genes of antibiotics. [Conclusion] The study demonstrates that diversity of endophytic actinobacteria from mangrove are rich and mangrove tissues as a special niche for discovery of new pharmaceutical microorganisms are waiting to be explored.

    • >Medical Microbiology
    • Inhibition of pathogenic Escherichia coli O1 and protection of infected mice by three Mongolian medicinal compounds

      2016, 43(8):1766-1773. DOI: 10.13344/j.microbiol.china.150657

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      Abstract:[Objective] We studied the antibacterial effect of three Mongolian medicinal compounds against pathogenic Escherichia coli O1 and their protection of mice infected with E. coli O1. [Methods] The effects of Mongolian medicinal compounds on the growth curve, cell surface hydrophobicity, the cytoderm and cell membrane permeability of E. coli O1 were determined by turbidimetry, the microbial adhesion to solvents method, reagent kit and membrane permeability assay with ONPG as a substrate. In addition, the protection of mice was evaluated by E. coli O1 challenge. [Results] All three compound recipes inhibited the growth of pathogenic E. coli O1 and protected mice against E. coli O1. The highest protection rate (50%) was obtained for mice treated with Mongolian medicinal compound recipe III, as same rate as ciprofloxacin treated mice. When acting on the E. coli O1 cells, the decoction of Mongolian medicinal compound recipe III consisted of 7 herbs firstly changed the surface hydrophobicity of the cell and secondly destroyed the integrity of the cell wall over time, and then destroyed the cell membrane and changed the permeability, which eventually destroyed the cell structure. [Conclusion] Mongolian medicinal compound recipe III showed the best antibacterial effect against E. coli O1 both in vivo and in vitro and its protection rate in mice infected with E. coli O1 was comparable with the antibiotic ciprofloxacin.

    • Inhibition of Cronobacter sakazakii biofilms by baicalin

      2016, 43(8):1774-1784. DOI: 10.13344/j.microbiol.china.150644

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      Abstract:[Objective] We studied the inhibition of Cronobacter sakazakii biofilm by baicalin. [Methods] XTT reduction assay was used to evaluate the effects of baicalin on the early adhesion of C. sakazakii planktonic cells and the activity of sessile cells in biofilms. Quantitative real-time PCR (qRT-PCR) was used to determine the expression level of the biofilm-associated genes, including glpQ, nlpD, gsiB, deoB, luxS, and sdiA. [Results] The inhibition of C. sakazakii biofilms by baicalin was dose dependent. MIC80 of baicalin against C. sakazakii was 1 024 mg/L, with 83.7% inhibition of C. sakazakii ATCC BAA-894 and 53.2% of C. sakazakii IQCC10423. In addition, 2 048 mg/L of baicalin inhibited the adherence of C. sakazakii at its earlier development of new biofilms. qRT-PCR results demonstrated that baicalin could down-regulate the expression of biofilm-associated genes in C. sakazakii and further inhibited its biofilm formation. [Conclusion] Baicalin could be potentially used as an antimicrobial to inhibit C. sakazakii biofilms.

    • >COMMUNICATIONS
    • Screening of low-temperature fermentation lactic acid bacteria from silage of Elymus nutans growing on the Qinghai-Tibetan Plateau

      2016, 43(8):1785-1794. DOI: 10.13344/j.microbiol.china.150611

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      Abstract:[Objective] This study is aimed to investigate lactic acid bacteria diversity in Elymus nutans silage, and selected out lactic acid bacteria which grew well at low temperatures of 10, 15 and 25 °C. [Methods] Lactic acid bacteria (LAB) strains isolated from Elymus nutans silages were identified by 16S rRNA gene sequences. Meanwhile MRS medium was used to choose high absorbance LAB from each species at different temperatures (10, 15 and 25 °C), and green juice fermentation was used to get the low PH value one as the dominant LAB at different temperatures (10, 15 and 25 °C). [Results] A total of 108 LAB strains were isolated from a variety of stages of fermentation at different temperatures. Based on 16S rRNA gene sequence analysis and phylogenetic tree, these 108 strains were classified into 6 genera and 18 species. In combination with the screening results of culture medium and fermented green juice, we found that LS-24 showed obvious growth advantage at 15 °C (P<0.05), and PP-63 grew faster in the early stage of fermentation, while LP-21 made pH value dropped to 3.9 at 15 °C and had the maximum number of viable cellseory. [Conclusion] The lactic acid bacteria found in this experiment covered almost all common lactic acid bacteria genera found in silages, but species were fewer as compared to the reported lactic acid bacteria from silages. LP-21, LS-24 and PP-63 show a good growth and fermentation characteristics, therefore, these three strains were selected as preferred candidates of lactic acid bacteria strains for low temperature fermentation of ensiling forage.

    • Prediction and identification of B-cell epitopes of outer membrane protein Tp92 on Treponema pallidum

      2016, 43(8):1795-1799. DOI: 10.13344/j.microbiol.china.150624

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      Abstract:[Objective] To predict and identify the B-cell epitopes of outer membrane protein Tp92 on Treponema pallidum and provide the basis for further discussion on effects of these epitopes for the epitope-based syphilis vaccine. [Methods] The B-cell epitopes of the protein Tp92 was analyzed with comprehensive meta-analysis Mobyle, ABCpred and IEDB online softwares, and the six peptides containing predicted epitopes were artificially synthesized. The sera from syphilis patients and Tp92-immunized rabbits (normal human and normal rabbits as negative control) were used to determine the immunoreactivity and specificity of six predicted peptides of Tp92 by indirect ELISA. [Results] Comprehensive meta-analysis of online softwares showed that P1 (24-39AA), P2 (332-347AA), P3 (520-536AA), P4 (575-588AA), P5 (103-118AA), and P6 (694-712AA) might be the B-cell epitopes. The result of indirect ELISA indicated that P1、P3、P5 and P6 were active with syphilis patient sera and Tp92-immunized rabbit sera but not with negative control sera. [Conclusion] These results indicate that P1、P3 、P5 and P6 are the potential specific B-cell epitopes, and immunoreactivities of P3 and P6 are the strongest especially.

    • >REVIEWS
    • Research progress of bacterial sesquiterpenoid biosynthesis

      2016, 43(8):1800-1813. DOI: 10.13344/j.microbiol.china.150606

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      Abstract:Terpenoids are a diverse group of natural products and play important roles in all living organisms. Terpenoid biosynthesis has been extensively studied in plants and fungi. However, cloning and engineering terpenoid pathways in these eukaryotic organisms remain challenging. Terpenoids are also produced by numerous bacteria. The research progress in bacterial terpenoid synthesis for decades made significant contributions to our understanding of terpenoid biosynthesis. Here we will focus on bacterial sesquiterpenoids and briefly review their chemical structures, the mechanisms of farnesyl diphosphate cyclization catalyzed by sesquiterpene cyclases, post modifications of sesquiterpenes by enzymes especially oxidoreductases, metabolic regulations and unsolved problems in biosynthetic pathways.

    • Advances in selection markers and their bio-safety in applications of transformed microorganisms

      2016, 43(8):1814-1821. DOI: 10.13344/j.microbiol.china.150618

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      Abstract:Selection markers are one of the basic parts in a genetic transformation system in molecular biotechnology. The functions of selectable marker genes are selecting transformants from non-transformed cells and maintaining a stress for selective growth of recombinant population. Drug-resistant genes are the most commonly used selectable marker genes, usually as a part of Escherichia coli vectors and other shuttle-vectors. It is considered bio-safe to use engineered strains carrying drug-resistant genes in fermentations of enzymes or organic compounds, because the fermentor systems are closely controlled and the products are to be refined. But the application of drug-resistant genes should be prohibited when genetic modification of the strains to be used for food/feed fermentation, environment remediation, plant bio-protection, and so on. Therefore, the development of bio-safety selection markers has been a key issue in the application of molecular biotechnology. This paper reviews the types and properties of currently used selection markers, and introduces the novelty and advantages of selection marker GFAT, a gene coding for a glucosamine synthetase. Selection marker GFAT will be useful in natural environments because no need to add or delete any compounds for generating selection stress.

    • Research progress on interaction between bacterial sRNA and quorum sensing system

      2016, 43(8):1822-1828. DOI: 10.13344/j.microbiol.china.150725

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      Abstract:Bacterial sRNA is a class of noncoding RNA with 40?500 nucleotides in length. sRNA plays important roles in regulating gene expressions in response to diverse stresses in the environment. In this paper, we reviewed the progress of the research on the interaction between bacterial sRNA and quorum sensing system, which may be of great significance in revealing the intricate metabolic processes of bacteria, as well as understanding the mechanism of bacterial response to changes in the environment.

    • Research advances in O-antigen polysaccharide of Salmonella enterica

      2016, 43(8):1829-1835. DOI: 10.13344/j.microbiol.china.150804

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      Abstract:O-antigen (O-polysaccharide) is a part of the lipopolysaccharide component of the outer membrane of Salmonella and consists of oligosaccharide repeats (O units). It is one of the most variable cell constituents and the diversity is a common basis for bacterial serotyping. Genes, which are responsible for O-polysaccharide synthesis and translocation, exist in Salmonella genome into cluster, called O-antigen gene cluster. The presence of O antigen plays a role in bacterial virulence and is essential for invasion, survival and colonization of bacteria in their natural environment. Besides, O-antigen is also a major protective antigen of Salmonella, and it can stimulate the host to produce strong immune responses, which contribute to bacteria elimination. Thus, it has extensively been targeted for vaccine development. This review summarizes O-antigen in terms of gene structures, synthesis, function and its application in vaccine development.

    • Advances of species diversity of arbuscular mycorrhizal fungi

      2016, 43(8):1836-1843. DOI: 10.13344/j.microbiol.china.150627

      Abstract (1653) HTML (1067) PDF 457.96 K (3763) Comment (0) Favorites

      Abstract:Arbuscular mycorrhizal fungi play important roles in different ecosystems of the world, to understand the species diversity of AMF could provide scientific basics for conservation and utilization of species resources, while the unculturable nature and higher genetic variability of AMF severely hampered the further advances of AMF. With the developing of research methods and application of the next-generation sequencing technology, it could reveal deeper insights into the AMF species diversity. This paper reviewed the advances in AMF classification systems, AMF species diversity in different host plants and habitats, and research methods (including morphological identification, Sanger sequencing and high throughput sequencing) of AMF species diversity and main problems that occurred in the study of AMF species diversity were also discussed. It was considered that not only new research technologies should be applied to study AMF species diversity in the future, but also the problem of unculturable nature of AMF should be concerned.

    • Distribution, spread and detection methods of antibiotic resistance genes in livestock manure and soil system

      2016, 43(8):1844-1853. DOI: 10.13344/j.microbiol.china.150872

      Abstract (1583) HTML (1222) PDF 503.40 K (2973) Comment (0) Favorites

      Abstract:There are increasing concerns about antibiotic resistance genes (ARGs) as emerging environmental contaminants. Antibiotics have been routinely utilized in livestock farming as feed additives to promote animal growth, prevent and treat diseases caused by various bacteria pathogens. Most of them could not be totally absorbed by animals but induce the development of antibiotic resistance bacteria (ARB) and ARGs in animal intestinal tracts. The residual antibiotics, ARB or ARGs are discharged with fecal. Therefore, animal wastes are important reservoirs of antibiotics, ARB, and ARGs. Antibiotic resistance may be transferred to the soil environment through composting and manure fertilization process. Furthermore, ARGs can disseminate in the environment by horizontal gene transfer, and even spread along the food chain, which poses a huge threat to the environment and human health. In this paper, the profiles of the occurrence, distribution and spread of ARGs in animal manure-soil system are reviewed in detail. The main detection methods of antibiotic resistance are discussed. Composting is still an effective way to reduce the spread of antibiotic resistance genes in animal manure and therefore should be an important consideration in the future.

    • >EDUCATION
    • Teaching reforms the systematicness and comprehensiveness of Applied Bioinformatics

      2016, 43(8):1854-1862. DOI: 10.13344/j.microbiol.china.160335

      Abstract (1783) HTML (986) PDF 2.58 M (2334) Comment (0) Favorites

      Abstract:Facing huge information, the traditional lecturing mode encountered lots of problems regarding knowledge fragmentation and plainness, which makes study boring. Applied Bioinformatics is an important tool for connecting knowledge points and can be utilized to solve the problems. To utilize well, this paper discussed the systematic and comprehensive reforms of the Applied Bioinformatics Course. This paper illustrated the teaching reforms that covered two aspects: the systematic outline of teaching contents and the comprehensive analysis of biological topics from popular science video. Based on feedbacks, students benefit a lot from the reforms and many aspects of their qualities were promoted. In the current information age, life science has been changing from the traditional experimental science to the rational design and experimental verification science, which dominated by thinking and analyzing. The Applied Bioinformatics course plays an important role in training such kind of research abilities. The systematic and comprehensive teaching reforms have made certain achievements, which has worth of learning and communicating.

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