• Volume 40,Issue 2,2013 Table of Contents
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    • >NEWS AND VIEWS
    • Biological control and growth-promoting microbes being paid attentions in agriculture microbiological research China

      2013, 40(2):211-211.

      Abstract (1454) HTML (0) PDF 145.03 K (2769) Comment (0) Favorites

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    • >Commentary
    • Potential value of thermostable cellulase-producing fungi in organic fertilizer production

      2013, 40(2):380-380.

      Abstract (1476) HTML (0) PDF 127.13 K (2648) Comment (0) Favorites

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    • >Industrial Microbiology
    • Optimization of fermentation conditions of recombinant β-xylosidase extracellularly expressed in E. coli

      2013, 40(2):212-219.

      Abstract (2147) HTML (0) PDF 552.01 K (4550) Comment (0) Favorites

      Abstract:[Objective] The fermentation conditions for production of recombinant β-xylosidase from Paecilomyces thermophila expressed in E. coli were optimized and produced in a 5 L fermentor. [Methods] Single factor experiments were used to investigate the effect of different inducers and their concentrations, initial cell density, cultivation temperature and time on the expression of recombinant β-xylosidase. [Results] The results showed that the β-xylosidase could be induced by IPTG and lactose, lactose was better than that of IPTG. Under the optimal conditions by adding 2% lactose at a initiated OD600 of 0.8?0.9 and incubation for 48 h at 33 °C, the ratio of extracellular enzyme reached over 99% and enzyme activity was 103.9 U/mL. Further optimization of the culture conditions were performed in 5 L fermentor, the highest β-xylosidase activity of 392.5 U/mL and extracellular protein concentration of 10.1?g/L were obtained after 48 h. [Conclusion] The recombinant β-xylosidase with high level production will be useful in various industrial applications.

    • >Environmental Microbiology
    • Isolation and identification of a cellulose degrading fungi

      2013, 40(2):220-227.

      Abstract (1875) HTML (0) PDF 520.80 K (4875) Comment (0) Favorites

      Abstract:[Objective] The aim of this study was to screen efficient cellulose degrading strains and to investigate their cellulase-producing conditions. [Methods] The fungus were screened from sugarcane soil by using the Congo red dye test, then measured the degradation rate of filter paper and the enzyme activity for second-screening. [Results] The hydrolyzation zone, clearing zone ratios (clearing zone diameter/colony diameter), the degradation rate of filter paper and cellulose activity were taken as evaluating selection criteria. We isolated one fungi with cellulose degrading ability, of which SJ1 was identified as Penicillium oxalicum by morphological and molecular technology. The results showed that the filter paper activity (FPA), endoglucanase activity (CMCase activity), β-glucosidase activity and exoglucanase activity (CBH activity) of SJ1 were 25.15 U/mL, 740.42 U/mL, 58.03 U/mL and 2.442 U/mL, respectively. [Conclusion] Strain SJ1 was a cellulase-producing strain with broad development potential.

    • >Agricultural Microbiology
    • Inhibitory of Bacillus vallismortis to Trichoderma parasitized in Lentinus edodes

      2013, 40(2):228-235.

      Abstract (1860) HTML (0) PDF 573.64 K (3754) Comment (0) Favorites

      Abstract:[Objective] The inhibition of Bacillus vallismortis to Trichoderma was researched in this article, because Trichoderma was a harmful bacterial with a higher infection rate in the mushroom production and was normally killed by chemical agent with the result of pesticide residues. [Methods] Firstly, the components of Bacillus vallismortis were confirmed if they were the intracellular or extracellular materials. Then the stability of the physical and chemical properties of the effective component was analyzed after extraction. Finally, initial application in the mushroom cultivation was probed. [Results] The lipopeptide extracted from exocellular secretion of Bacillus vallismortis can effectively inhibit the germination of spores and mycelial growth. The inhibition rate could reached 62.8% with 0.02 g/L, little influence on the growth of mushroom mycelium, relatively stable physical and chemical properties. An addition of 26.6% 36 h fermentation broth of Bacillus vallismortis in the mushroom cultivation compound had the optimum inhibition on Trichoderma hyphae. [Conclusion] Extracellular secretions of Bacillus vallismortis had good inhibition on Trichoderma. It was worth further investigating considering the reduction of pesticide residues, though the mushroom mycelium germination was also inhibited at a certain degree.

    • Detection of sweet potato viruses in Yunnan and genetic diversity analysis of the common viruses

      2013, 40(2):236-248.

      Abstract (2213) HTML (0) PDF 641.70 K (4143) Comment (0) Favorites

      Abstract:[Objective] To determine the sweet potato virus species in Yunnan and analyze the genetic diversity of the main viruses. [Methods] Two hundred seventy-nine sweet potato samples with virus-like symptoms collected from 16 counties/cities in Yunnan Province were tested by RT-PCR/PCR using virus-specific primers for 8 sweet potato viruses. The RT-PCR amplicons of SPFMV, SPVC and SPVG from selected samples were cloned and sequenced. The alignments and phylogenetic analyses were performed with the MEGA 5 software package. [Results] Six viruses, sweet potato chlorotic fleck virus (SPCFV), sweet potato feathery mottle virus (SPFMV), sweet potato leaf curl virus (SPLCV), sweet potato virus C (SPVC), sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2), were detected in 123 samples (44.1%) from 14 counties/cities except Puer and Xianyun. Of all the detected viruses, SPVG was detected in 109 samples (39.1%), SPFMV in 75 samples (26.9%), SPVC in 69 samples (24.7%), and SPLCV just in one sample (0.4%). Mixed infections of 2–5 viruses were common (31.90%) in Yunnan. Among the mixed infections, most of them were caused by 2–3 viruses. Only 34 samples (12.2%) were caused by one virus, however, no single infection was detected for SPCFV, SPLCV and SPV2. [Conclusion] Sequenced analysis showed that the Yunnan isolates of SPFMV belonged to three strains, EA strain, O strain, and another strain different from EA, O and RC. Both SPVC and SPVG isolates were divided into three groups, and most of the Yunnan SPVG isolates were in the group I.

    • Establishment of molecular ID in 11 Ganoderma lucidum strains

      2013, 40(2):249-255.

      Abstract (1612) HTML (0) PDF 444.81 K (3445) Comment (0) Favorites

      Abstract:[Objective] Eleven Ganoderma lucidum strains were collected as materials for classifying them at the molecular level and establishing the molecular ID. [Methods] Internal transcribed spacer (ITS) and simple sequence repeat (SSR) markers were used for the molecular identification of eleven Ganoderma lucidum strains. [Results] 99% similarity in ITS sequence between the tested strains and the Ganoderma lucidum registered in GenBank, meaning that the tested strains were Ganoderma lucidum species. The cluster analysis by NTSYS revealed that eleven Ganoderma lucidum strains were divided into four groups at similarity coefficients of 0.62. GL-2 and GL-4 were in two clades respectively. According to fragment size of allele variation, the agarose gel electrophoresis bands were analyzed by the software ID Analysis 1.0. Five primer pairs could be used to identify all the tested strains and accomplish the establishment of molecular ID. [Conclusion] Establishment of molecular ID in Ganoderma lucidum based on SSR were feasibly.

    • >Food Microbiology
    • Damaging effect of pressurized CO2 on cell membrane of Escherichia coli

      2013, 40(2):256-265.

      Abstract (2170) HTML (0) PDF 938.94 K (3695) Comment (0) Favorites

      Abstract:[Objective] The damage in the cell membrane of bacteria could be reflected on the leakiness of the material in bacteria and the absorption of dye in bacteria. Compared with pasteurization (63 °C, 30 min), the damaging effect of pressurized CO2 on the cell membrane of Escherichia coli was studied. The aim of the study was to analyze the relationship between the death of Escherichia coli and the damage of cell membrane. [Methods] The change of cell membrane permeability and the leakiness of protein and nucleic acid in Escherichia coli were detected. The change of Escherichia coli morphology was observed by TEM. [Results] The results indicated that pressurized CO2 treatment induced the change of cell membrane permeability of Escherichia coli. Pressurized CO2 treatment induced the leakiness of protein in Escherichia coli, but the time of leakiness was lagged behind the time of 99% Escherichia coli death, so it was not the reason for death, it was only the secondary phenomenon of death. The death of Escherichia coli could be related to the leakiness of nucleic acid induced by the pressurized CO2 treatment. The death of Escherichia coli could be related to the ultrastructure change of Escherichia coli induced by the pressurized CO2 treatment. [Conclusion] There was direct relationship between the damaging effect of pressurized CO2 on cell membrane of Escherichia coli and the death of Escherichia coli.

    • >Medical Microbiology
    • Analysis of factors influencing the infectivity of HIV-1 pseudotyped virus during packaging by cell line

      2013, 40(2):266-273.

      Abstract (2037) HTML (0) PDF 568.75 K (6764) Comment (0) Favorites

      Abstract:[Objective] A serials of factors which may have influence on producing the HIV-1 pseudotyped virus are analysed to establish an optimal way to generate this type of virus. [Methods] A set of factors such as the transfection reagents, the ratios of plasmid and transfection, the serum in the media and cell cycle is being analysis to find the optimal condition. [Results] The optimal N/P is range from 8 to 40, the serum can influence the stability of pseudotype virus and in the stage S and G2/M, transfection efficiency is high than the stage G0/G1. [Conclusion] High titers of pseudotyped HIV-1 virus can be obtained by means of using transfection reagents PEI and through combinatorial optimization of PEI, plasmid amount and FBS type.

    • >COMMUNICATIONS
    • Influence of different induction methods on Agrobacterium tumefaciens-mediated transformation efficiency of Trichoderma

      2013, 40(2):274-278.

      Abstract (1755) HTML (0) PDF 305.27 K (3483) Comment (0) Favorites

      Abstract:[Objective] To determine the optimum induction method, different induction methods on Agrobacterium tumefaciens EHA105-mediated transformation system of Trichoderma harzianum T88 were investigated. [Methods] By comparing the conversion efficiency of double cultivation, blotting cultivation and liquid co-cultivation, the optimum induction method can be determined. [Results] The conversion efficiency of double cultivation is about 10 transformants per 107 conidia, the conversion efficiency of blotting cultivation is about 20 transformants per 107 conidia, while the conversion efficiency of liquid co-cultivation is about 100 transformants per 107 conidia. [Conclusion] The conversion efficiency of liquid co-cultivation is the maximum in A.tumefaciens-mediated transformation system of T. harzianum, it is about 5?10 times of double cultivation and blotting cultivation.

    • >REVIEWS
    • The research progress on adjuvant therapy of allergic disease by probiotics

      2013, 40(2):279-286.

      Abstract (1961) HTML (0) PDF 393.31 K (5288) Comment (0) Favorites

      Abstract:Probiotics are defined as live microorganisms that, when administered in adequate amount can confer a health benefit on the host and regulate the immune system. Accumulated evidence has been demonstrated the alleviation of allergic symptoms by probiotics was conducted via regulation of Th1/Th2 balance as well as the improvement of oral tolerance of the hosts. In this paper, the research progresses in last decade in the anti-allergic effect of probiotics in vitro tests, in experimental animals as well as clinical trials are reviewed. It is shown probiotics is of great potentials in adjuvant treatment of allergic diseases.

    • The roles of polyamines on pathogenesis of bacterial pathogens

      2013, 40(2):287-293.

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      Abstract:Polyamines are aliphatic compounds with two or more amino groups. The intracellular content of polyamines is regulated by the biosynthesis, transport, degradation and interconversion processes. The polyamines and their biosynthesis and transport mechanisms have important roles on the pathogenesis of bacterial pathogens. This review focuses on the roles of polyamines on the transcription and translation of virulence factors of bacteria, the formation of biofilm, the resistance to antibiotics, the resistance to the acid stress and oxidative stress in host, the biosynthesis of molecules involved in pathogenesis and the resistance to the host.

    • Review on biosynthetic pathway of secondary metabolites and the related genes in Monascus spp.

      2013, 40(2):294-303.

      Abstract (2688) HTML (0) PDF 654.23 K (6153) Comment (0) Favorites

      Abstract:Monascus spp., one of the important microbial resources both for food and medicine in China, can produce kinds of useful secondary metabolites, such as the natural food additive Monascus pigments, the cholesterol-lowering substance monacolin K and so on. Meanwhile, this genus also has the ability to secrete the mycotoxin citrinin, causing a safety risk to Monascus-related products. Therefore, how to promote the production of the useful metabolites and eliminate the production of citrinin has become a hot topic. Over the past decade, with the development and application of molecular biological approaches in Monascus spp., great efforts have been made to explore the basical knowledge about biosynthesis of secondary metabolites, and many genes involved in biosynthesis of Monascus pigments, monacolin K and citrinin have been identified and characterized. The latest achievements were summarized in this paper, in the purpose of providing potential approaches to manipulate and improve industrial Monascus strains efficiently.

    • The progress of studies on a unique carbon dioxide fixation pathway: 3-hydroxypropionate cycle

      2013, 40(2):304-315.

      Abstract (2903) HTML (0) PDF 503.74 K (6306) Comment (0) Favorites

      Abstract:A novel carbon dioxide fixation pathway, 3-hydroxypropionate cycle, has been discovered in thermophilic filamentous photosynthetic bacteria such as Chloroflexus aurantiacus, which can grow in several conditions. The characteristic of this pathway is 3-hydroxypropionate as a metabolic intermediate, which can be used for synthesis of many valuable products in industry. In this minireview, the discovery of this unique biosynthetic process, the development of the reaction mechanism and the latest progress are introduced, especially the involvement of several multifunction enzymes. Besides theoretical significance, potential biotechnological applications of the 3-hydroxypropionate pathway are also discussed.

    • >EDUCATION
    • Reform and practice of microbiology experiment teaching in local normal university base on the vocation education

      2013, 40(2):316-321.

      Abstract (1744) HTML (0) PDF 345.10 K (3468) Comment (0) Favorites

      Abstract:According to the specialty aim of local normal university and the microbiology experiment teaching under the new curriculum reform of middle school, the microbiology experiment teaching in local normal university has been reformed considering the occupation ability training. In order to train the practical ability of students and develop students’ creative thinking, a set of innovation teaching system on microbiology experiment course to suit the biological science undergraduate in local normal university was to be established from the reform of the experimental content, teaching method, assessment system, students’ graduation thesis, and so on.

    • Construction of three-dimensional teaching system in food microbiology experimental teaching

      2013, 40(2):322-327.

      Abstract (1866) HTML (0) PDF 330.44 K (3261) Comment (0) Favorites

      Abstract:We reformed teaching contents, teaching means, teaching methods and evaluation methods of food microbiology experimental teaching in order to construct three-dimensional experimental teaching system which contained Basic experiment—Integrated improve experiment—Innovative experiment, so that it could possess systematic teaching, hierarchical courses and multiform contents. And it had trained the students well and improved their special skills. Good effects had been made since this system was very helpful to improve their consciousness of innovation and integrative abilities in doing experiments.

    • Reform and practice of the undergraduate research ability cultivation and the excellent academic discipline construction

      2013, 40(2):328-333.

      Abstract (1889) HTML (0) PDF 349.22 K (5053) Comment (0) Favorites

      Abstract:The university students’ capability of researching and creative talents should teach different students in different way, and in different term. Standardize training methods make students to use what they have learnt. Construction of fine style of study and encourage the students to study independently and honestly the inevitable request to improve the quality of education and teaching, cultivate high-quality talents, promote the sustainable development of university. Many universitys implemented undergraduate tutorial system in order to make the students study more individual and develop the potential ability of researching and creative talents in various universitys. The undergraduate researching and creative ability and the research tutorial system was defined and traced to the source. The students’ capability of researching and creative talents should be accorded with the needs of the times development. The researching and creative movement, combined with course teaching, experimental teaching, thesis design practice, undergraduate teaching plan, was advantageous to cultivate the comprehensive-quality and group cooperative spirit of undergraduate students. The students were principal part of construction of fine style, whereas the teachers were guiding.

    • Comprehensively construct the modern microbiology teaching system with the guideline of textbook research and compilation

      2013, 40(2):334-340.

      Abstract (1908) HTML (0) PDF 414.29 K (3528) Comment (0) Favorites

      Abstract:Supported by several national construction projects and based on the original foundation, we constructed a modern microbiology teaching system comprehensively with the guideline of textbook research and compilation. We produced a high level, full-scale textbook series with our own characteristics. We developed a powerful teaching team with members of different ages and all members pay the same attention to teaching and academic research. We also established a modern teaching method and system, while a pattern of “research learning” was created. Our effort promoted the national microbiology teaching level.

    • >BIOLOGICAL LAB
    • Detection of soft-shelled turtle iridovirus with HRCA

      2013, 40(2):341-349.

      Abstract (1989) HTML (0) PDF 494.10 K (3770) Comment (0) Favorites

      Abstract:[Objective] To establish a detection system of soft-shelled turtle iridovirus (STIV, a causative agent of severe diseases in cultured Trionyx sinensis) with hyper-branched rolling cycle amplification (HRCA) assay, which provides a stable, reliable and novel technique for early diagnosis of the disease and identification of the pathogen. [Methods] The padlock probe consists of a universal linking sequence and the two target complementary regions at 5′ and 3′ ends, which was designed based on the unique fragment sequence of STIV, was synthesized. Detection system of HRCA was established and optimized. The specificity and sensitivity of HRCA was determined and compared with conventional PCR. Sick turtles with or without obvious symptom were detected applying HRCA. [Results] HRCA is capable of amplifying STIV DNA, while can not detect related virus and other aquatic animal virus. The detection sensitivity of HRCA is as low as 101 copy which is 100-fold higher than that of conventional PCR. All the detections of the infected soft-shell turtles with or yet without evident symptoms at early stage of infection showed positive results. [Conclusion] The detection system of HRCA for STIV was successfully established. It is sensitive, specific, rapid and simple. It can be used for early diagnosis with good prospects for generalizing in the turtle culture.

    • Construction of high-level expression vector for Bacillus thuringiensis

      2013, 40(2):350-361.

      Abstract (2059) HTML (0) PDF 648.66 K (4272) Comment (0) Favorites

      Abstract:[Objective] To construct a vector for high-level expression of cry genes in Bacillus thuringiensis, a strong promoter was screened by comparing the transcriptional activities among cry1A, cry3A, cry4A and cry8E promoters. [Methods] The transcriptional activities of the four promoters were detected by fusing with lacZ gene. Scanning electron microscopy was used to detect the abilities of crystal protein formation. SDS-PAGE, protein quantitation and bioassay were performed for the functional verification of the high-level expression vector. [Results] The four promoters Pcry1A, Pcry3A, Pcry4A and Pcry8E were fused with lacZ report gene. Beta-galactosidase assay showed that the activities of the four promoters were, in decreasing order, Pcry8E>Pcry1A>Pcry4A>Pcry3A. The cry8E promoter was selected to construct the high-level expression vector pHT315-8E21b based on plasmid pHT315. cry1Ac gene was inserted into both pHT315-8E21b and the widely used plasmid pSXY-422b which was initiated by cry3A gene promoter, respectively. The two expression plasmids were introduced into the acrystalliferous mutants HD-73?, and HD-8E1Ac and HD-422-1Ac strains were obtained. HD-8E1Ac and HD-422-1Ac strains Cry1Ac protein was observed by scanning electron microscopy, the results indicated that HD-8E1Ac strain produce diamond crystals compared with HD-73?. SDS-PAGE and protein quantitation analysis showed that the expression of cry1Ac gene is significantly increased in HD-8E1Ac compared with that in HD-422-1Ac. Bioassay results showed that HD-8E1Ac possesses much higher toxic against Plutella xylostella compared with HD-422-1Ac. [Conclusion] The high-level expression vector pHT315-8E21b initiated by cry8E gene promoter has the ability to express Cry1Ac correctly. It has higher efficiency of expressing insecticidal crystal protein compared with the widely used plasmid pSXY-422b.

    • The sectionalized DNA shuffling: an effective tool for molecular directed evolution of Meiothermus ruber TreS

      2013, 40(2):362-372.

      Abstract (1954) HTML (0) PDF 832.88 K (3389) Comment (0) Favorites

      Abstract:[Objective] The gene M-treS from Meiothermus ruber CBS-01 encodes a trehalose synthase of 962 amino acids, named M-TreS. To improve its catalytic activity, we constructed a method of molecular directed evolution, the sectionalized DNA shuffling. [Methods] Through two PCR steps with two pairs of partially complementary primers, the M-treS gene was parted into two sections. After the two sections shuffled respectively, a whole gene was obtained through the complementarity of the primers. This method was more feasible, with higher mutability than normal DNA shuffling. [Results] Mutants were obtained after one round of the sectionalized DNA shuffling, in combination with error-prone PCR. The best mutant enzyme contained 6 amino acid substitutions, whose catalytic activity and efficiency were 1.6-fold and 2-fold of that of the wild type, respectively. In the 6 amino acid substitutions, 5 were caused by homologous recombination, and one by error-prone PCR. [Conclusion] This study indicates that the sectionalized DNA shuffling is an effective tool for molecular directed evolution of macromolecular proteins.

    • Rapid differentiation and identification of five species of Salmonella by FT-IR spectroscopy

      2013, 40(2):373-379.

      Abstract (2041) HTML (0) PDF 552.65 K (4066) Comment (0) Favorites

      Abstract:[Objective] To establish a standard fourier transform infrared (FT-IR) spectral library and a FT-IR method of differentiation and identification of five species of Salmonella, viz Salmonella typhimurium, Salmonella enteritidis, Salmonella choleraesuis, Salmonella arizonae and Salmonella potsdam. [Methods] FT-IR fingerprint absorption spectra of five species of Salmonella were collected and analyzed by chemometric methods. [Results] A standard FT-IR spectral library was created and could be used to compare with those of the target Salmonella and identify them; two cluster models of principal component analysis (PCA) and hierarchical cluster analysis (HCA) were established and could differentiate and identify well the five species of Salmonella. [Conclusion] As a rapid, easy-to-use and accurate technique, FT-IR spectroscopy is an effective tool to differentiate and identify five species of Salmonella studied here.

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