Abstract:

Abstract:[Objective] We tested an insect cells-based liquid medium and its utility for the easy-to-maintain, fast and reliable system for cultivation of different Bartonella. [Methods] Media composition and growth conditions were optimized using B. henselae and B. quintana. The growth curves of the other ten Bartonella species were determined using the optimized liquid medium. [Results] Glutamine and sucrose supplements did not affect significantly the growth rate of these bacteria. A generation time of 5.2 h for B. henselae and 4.3 h for B. quintana was estimated during the exponential phase of the growth curve occurred between days 1 and 2 after inoculation. The twelve medical and veterinary important Bartonella species grew well under conditions established and each exhibited unique growth characteristics. [Conclusion] This liquid growth medium may provide an advantage over conventional direct blood agar plating for analyzing Bartonella properties including antibiotic susceptibility and resistance and pathogenicity.

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Abstract:[Objective] Three different environmental samples were mixed and inoculated into 100 mL fresh medium containing 1% pyrolusite and 1% pyrite. After enrichment we got bioleaching microflora. [Methods] Obtained microorganism consortia were used to leach pyrite and low-grade pyrolusite. Non-inoculation system was conducted as control group. [Results] Series of parameter were monitored during the process, including dynamic of consortia construction, pH value, leaching ratio of manganese, and XRD analyze of leaching residual. Leaching ratio of manganese was 92.48% after 15 days cultivation. And it was much higher than control group (40.34%). Within microbial consortia, Thiomonas sp. was the dominant species and its proportion increased from 2% to 93% of the consortia. Value of pH was declined from 4.0 to 2.5 in experimental group. Jarosite was observed in bioleaching residual. [Conclusion] Decrease of pH value and formation of jarosite indicated abundant sulfuric acid produced by bacterial metabolism. Result suggested that increasing bioleaching ratio of pyrite and pyrolusite due to dissociation of pyrite and decline of pH value by bacterial activity. This study could provide the basis for the further research of bioleaching mechanism and improvement of industrial art of low-manganese ore mineral resource.

Abstract:[Objective] Maar lake is a special type of Crater Lake. Huguangyan Maar Lake was formed about 140 k?160 k years ago, fully closed, and has not yet been affected by human activities, where abundant and novel microbial species might dwell as reported previously. Aerobic anoxygenic phototrophic bacteria (AAPB) is a functional bacterial group with long evolution history in Earth possessing unique physiological and ecological characteristics. To date, our knowledge about AAPB distribution in Maar Lake is still blank. [Methods] Here, by constructing and analyzing six clone libraries of the photosynthetic reaction center pufM gene from total DNA and RNA, respectively, with 1 m, 5 m, and 12 m water layers in Huguangyan Maar Lake, and combining quantitative Real-time PCR, we studied AAPB’s distribution, phylogenetic diversity and the proportion in the total bacteria in different water layers. The results of coverage value and rarefaction curves of six libraries showed that AAPB diversity was sampled well for the purpose of revealing the diversity of main AAPB groups in each water layer. [Results] BLAST analysis showed that pufM sequences in Maar Lake were 80%?93% similar to public sequences. Diversity index indicated that the AAPB diversity in surface and deep layers was similar, whereas diversity in the intermediate layer was lowest. In view of total RNA and DNA data, pufM RNA diversity was higher than that of DNA. Phylogenetic and statistical analysis revealed that 49.43% sequences are fell into the OTUs 21?24 which were closely related to β-proteobacteria and represent dominated AAPB groups. Quantitative PCR results showed that the percentage of AAPB in total bacteria in 1 m water layer reached a highest value of 38.06%, whereas only 0.85% and 9.54% in 5 m and 12 m, respectively. [Conclusion] Huguangyan Maar Lake is occupied by rich and diverse AAPB groups.

Abstract:[Objective] Isolating and characterizing biosurfactant-producing bacteria. [Methods] Biosurfactant-producing bacteria were isolated by enrichment method. The morphological, biochemical and physiological characteristics and 16S rRNA gene sequence were determined to identify the taxonomic position of strain 3372. The optimal conditions for the growth and biosurfactant production of strain 3372 were investigated. [Results] Strain 3372 was isolated from marine water sample. It grows from pH 7.0 to 11.0 (optimum at pH 9.0), from 10 °C to 45 °C (optimum at 30 °C) and up to 12 % salinity. 16S rRNA gene sequence analysis showed that the strain belonged to the genus Dietzia and most closely related to Dietzia cercidiphylli. Strain 3372 was able to grow and reduce the surface tension of culture broth to 32.95 mN/m when cultured using paraffin as sole carbon source at pH 9.0 and 3% salinity. The biosurfactants produced by strain 3372 was identified as lipopeptides by thin layer chromatography. [Conclusion] Strain 3372 was a new alkaliphilic member of lipopeptides-producing bacteria, which has potential application in the processes where high pH is common.

Abstract:[Objective] The lipase gene of P. alcaligenes was cloned and expressed in Escherichia coli, the enzymatic properties were characterized. [Methods] The lipase gene was obtained by constructing the genomic library of P. alcaligenes and PCR. Then the gene overexpressed in E. coli BL21(DE3) with plasmid pET30a(+). The recombinant lipase was purified with HisTrapTM affinity chromatography and the enzymatic properties were determined. [Results] A 1 575 bp lipase gene was attained (GenBank assession number: JN674069). The molecular weight of lipase was 55 kD, the optimal substrate of the lipase was p-NPO, the optimal temperature and pH were 35 °C and pH 9.0. The lipase activity was increased to 156% after treated by 1 mmol/L Cu2+ for 30 min. Under the optimum conditions, the specific activity, Km and Vmax of the enzyme were 275 U/mg, 80 μmol/L and 290 mmol/(min·g protein), respectively. [Conclusion] The cloning and expressing of lipase provided the fundamental to the application in chiral resolution.

Abstract:[Objective] Acetolactate synthase (ALS) is the key enzyme in isobutanol biosynthetic pathway. Efficient expression of ALS is of great significance for the regulation of isobutanol metabolic pathway. [Methods] The acetolactate synthase gene (alsS) from Bacillus subtilis was amplified by PCR with primers designed according to the sequence of alsS in GeneBank, which is 1 713 bp. Then the alsS was cloned into the expression vector of pET-30a(+). The resulted recombinant plasmid was transformed into Escherichia coli BL2l(DE3) for the overexpression of alsS. [Results] The heterologous expression condition was optimized to be inducted at an OD600 of 0.6?0.8, 30 °C with 1 mmol/L IPTG for 6 h. ALS was mostly expressed in the supernatant with the activity of 24.4 U/mL, which was improved for 7.13 times. Electrophoretically pure ALS was obtained after HisTrapTMFF affinity chromatography with the specific activity of 95.2 U/mg. [Conclusion] These results contributed to the construction of isobutanol biosynthetic pathway in E. coli.

Abstract:[Objective] To express and purify human enterokinase light chain (hEKL) in prokaryotic expression system and detecting its activity in vitro. [Methods] The fragment of hEKL gene was amplified by PCR and cloned into plasmid pMAL-s downstream to the gene of fusion partner MBP-tag. The recombinant plasmid pMAL-s-hEKL was transformed into E. coli BL21(DE3). After induced expression, affinity chromatography was amplified to purify the target protein and Tricine SDS-PAGE was used to analyze the enzymatic activity. [Results] Majority of the fusion protein MBP-hEKL was expressed in soluble form. 40 mg protein was obtained with a purity of more than 97% from 1 L fermentation broth. Activity analysis showed that MBP-hEKL could cleavage the fusion protein with enterokinase recognition site specially and the specific activity was reached to 6.0×105 U/mmol.

Abstract:[Objective] Watermelon Fusarium wilt caused by Fusarium oxysporum f. sp. niveum is a common destructive soil-borne disease. Understanding of the competition between non-pathogenic congeneric Fusarium species and pathogenic Fusarium oxysporum strains has contributed to acquisition of novel bio-control agents and broadening the biological control measures against the plant disease. [Methods] Selective media and dilution plating procedure were used to study colonization of bulk soil, rhizosphere soil and plant tissues of watermelon grown in greenhouse pots by non-pathogenic Fusarium verticillioides XA and pathogenic Fusarium oxysporum LD. [Results] Strains XA and LD were isolated from diseased plant tissues in field. When non-diseased soil was solely inoculated with strain XA or strain LD, inoculation with strain XA had no symptom of wilt and no loss of biomass of watermelon whereas inoculation with strain LD led to heavily symptom of wilt. As compared with the treatment of LD, the treatment of dual inoculation with strains XA and LD increased the fresh weight and dry weight of the aerial part of watermelon plants by 151.2% and 110%, respectively. Strain XA successfully colonized roots but was not found in the basal part of stem of watermelon, while strain LD infected plant tissues and soil with (1.58?4.85)×104 CFU/g. As compared with the treatment of LD inoculation, the dual inoculation with strain XA and strain LD decreased the pathogenic F. oxysporum numbers in the basal part of stem, roots, rhizosphere soil and bulk soil of watermelon by 63.3%, 66.1%, 3.3% and 24.4%, respectively and increased the non-pathogenic F. oxysporum numbers in roots, rhizosphere soil and bulk soil of watermelon to (0.35?3.84)×104 CFU/g; this treatment gained 57.8% of control efficiency against watermelon Fusarium wilt. [Conclusion] The non-pathogenic congeneric strain XA effectively reduced the ability of pathogenic F. oxysporum LD to infect watermelon plants and had certain effectiveness as a biocontrol agent against watermelon Fusarium wilt.

Abstract:[Objective] The bacteria from gut of black soldier fly were screened gainst plant pathogenic bacteria, Molecular method was used to identify the active substances from the antagonist bacteria. [Methods] Eleven strains have been isolated with diluted coating method from the gut of black solider fly. The antagonistic strains were screened using the plate confrontation method. The species was identified through the physiological, biochemical experiments and 16S rRNA phylogenetic analysis. Primers of key genes of known lipopeptide, synthesis were designed and purpose fragements were amplified by PCR and sequenceing. [Results] A gut bacteria, named BSF-CL, was obtained with strong inhibitory effect to Xanthomonas oryzae PXo99 and Rhizoctonia solani AG-8, and identified as Bacillus subtilis. The result of PCR showed that strain BSF-CL possibly has the key genes which synthesis lipopeptide Iturin and Surfactin. We speculate that strain BSF-CL can synthesize lipopeptide iturin and surfactin. [Conclusion] A strong active Bacillus subtilis strain BSF-CL against bacterial and fungal pathogen was screened from the gut of black soldier fly. We preliminary speculated that the active substance may be lipopeptide iturin and surfactin through the molecular cloning and identification.

Abstract:[Objective] Screened bacterial strains which had a significant effect on mycelia and fruit body growth, and further made species identification. [Methods] Added the single strain of three bacterial strains C1, T24, T34 and four introduced bacterial strains 1B00263, 1A0081, 1A03263, 1A01082 to the casing layer, and then regular survey was mad, including the length of mycelia growth, time for primodia to emerge, number of primodia, and number and weight of mature fruit bodies. After the bacterial strains that had a significant effect on mycelia and fruit body growth were screened, molecular biology identification was further made. [Results] Adding T34 strain had a great effect on the fruit body. T34 strain not only could promote the growth of fruit bodies, but also played an important role in promoting fruit body formation and increasing the weight of individuals, indicating T34 was an ideal bacterial strain. Adding C1, T24, 1A01082, 1B00263 and 1A03263 had significant effects on fruit body growth. Meantime, adding 1A00081, 1B00263, C1 and 1A03263 could shorten the time for primodia to emerge (treatment 19.86?24.14 d and control 27.00 d). C1, T24 and T34, which had different effect degrees of on the growth of mycelia and fruit body, were further made molecular biology identification. The identification results were as follows: C1 belongs to Brevibacterium, T34 belongs to Bacillus, and T24 needs to be further determined. [Conclusion] By adding seven bacterial strains to the casing soil, we studied their effects on the growth of mycelia and fruiting body of casing soil Phlebopus portentosus. In result T34 (Bacillus sp.) was screened as the one bacterial strain that had a significant promotional effect on both mycelia and fruit body growth.

Abstract:[Objective] The purpose of this study was to isolate a butanol-producing strain from the soil of corn field in Shiquan County, Shaanxi Province, China, and to improve its butanol tolerance and butanol yield. [Methods] Using the multi-factor complex screening and the treatment of butanol stress acclimation, the strain with a high yield and butanol tolerance was obtained. [Results] The results showed that through several batches of acclimation and screening, the mutant T64 was derived from isolated wild-type strain D64, its butanol tolerance was significantly improved and could grow well in the complex screening medium containing 20 g butanol/L. It produced 21.8 g/L total solvent (acetone, butanol, ethanol) and a butanol yield reached 15.18 g/L using 7% corn mash as fermentation medium, which was higher than that of the wild-type strain D64 (13.35 g/L). [Conclusion] In conclusion, the designed multi-factor complex screening is more effective than the mono-factor screening for obtaining high-producing butanol strain. In addition, the stress acclimation of increasing butanol concentration in long term provides new ideas to research the tolerance of butanol.

Abstract:[Objective] Investigation of occurrence of Streptomyces linear and circular plasmids in some extremely natural habitats. [Methods] Twenty soil samples from the Tibet plateau of China were collected, Streptomyces strains were identified and plasmids were isolated. [Results] Forty-six Streptomyces strains were obtained and, surprisingly, half of them harbored from one to four linear plasmids of 19–650 kb, and eight strains contained from one to four circular plasmids of 4–80 kb. [Conclusion] The abundance and diversity of linear and circular plasmids in Streptomyces strains from Tibet suggests that the extreme environmental stress, such as highly solar radiation, might induce DNA damage and repair to promote formation of varied plasmids.

Abstract:[Objective] A high-yield γ-aminobutyric acid (GABA) producing lactic acid bacterium (LAB), strain M1 was isolated from pickled Chinese vegetables by our laboratory. [Methods] Its physiological and biochemical characteristics and 16S rDNA sequence were analyzed. GABA fermentation medium, based on MRS medium, was optimized using single factor test and orthogonal design, as well as the shake flask condition for GABA fermentation. [Results] The results indicated that the morphological, physiological and biochemical characteristics of strain M1 were accorded with Enterococcus family. The identity between the obtained 16S rDNA sequence of strain M1 and Enterococcus raffinosus SS1278 was up to 99%, strain M1 was accordingly identified as Enterococcus raffinosus. Moreover, its GABA fermentation medium was optimized, and the shake flask condition for GABA fermentation was: inoculum of 10%, growing temperature at 30 °C, initial pH at 5.5, fermentation period of 60 h and monosodium glutamate substrate concentration of 10%. Under these conditions, GABA yield by flask fermentation achieved a 1.22-fold increase. [Conclusion] The isolated E. raffinosus M1 strain reveals its potential for industrial fermentation of GABA.

Abstract:[Objective] To identify the three jujube witches’-broom phytoplasmas strains from Tai’an. [Methods] The phytoplasmas strains were identified by 16S rDNA sequence analysis via PCR amplification of 16S rDNA gene with the universal primer pair of R16F2n/R16R2 for phytoplasmas and online virtual 16S rDNA-RFLP analysis, and then their 16S rDNA sequences were deposited in GenBank. [Results] The results showed that the 16S rDNA sequences of the three strains shared the identity of 99.5%?99.7% with that of jujube witches’-broom phytoplasmas (AB052876 and AF279272), cherry lethal yellows phytoplasmas (AY197659) and apricot leaf roll phytoplasmas (FJ572660) which belong to 16Sr Ⅴ-B subgroup, and they were named as jujube witches’-broom phytoplasma strain Yuanling1 (JWB-Yuanling1, TA), jujube witches’-broom phytoplasma strain Lubeidongzao (JWB-Lubeidongzao, TA) and jujube witches’-broom phytoplasma strain Dabailing (JWB-Dabailing, TA) respectively. The 16S rDNA sequences of three JWB strains were deposited in GenBank with the accession numbers of HM989946, HM989947 and HM989948 respectively. [Conclusion] The three phytoplasmas strains from Tai’an were identified as the members of 16Sr Ⅴ-B subgroup.

Abstract:Firefly luciferase is the key component of ATP bioluminescence reagent, gained from firefly lantern throuh extraction and purification or preparation through genetic engineering, the performance of ATP bioluminescence reagent was decided by the vitality and the purity of firefly luciferase. Up to now, many present advanced technology were applied on preparation the reagent such as genetic engineering, ATP amplification device, stabilization technology of luciferase protein and luminescence, and so on. Now research focus on improving detection sensitivity and luminescence performance of the ATP bioluminescence reagents, further raising the adaptability of ATP bioluminescence reagents.

Abstract:Hexachlorocyclohexane (HCH) is a halogenated organic insecticide, which was once extensively used all over the world for agricultural and public health purposes. Its use has been restricted or banned in the developed countries due to its high toxicity and long persistence. However, HCH is still used in some developing countries. Even in those countries, where the use of HCH has been stopped for many years, the problem of residues still existed. In this article, we summarized the latest progress in the research of the diversity of HCH-degrading microorganisms and the degradation pathway on the four main HCH-isomers (α-, β-, γ- and δ-HCH), which will provide the reference for the economical and feasible bioremediation strategy on the HCH polluted environment.

Abstract:Shewanella oneidensis MR-1, a model dissimilatory metal-reducing bacterium, can effectively utilize many metal compounds and artificial synthetic dyes as electron acceptors to anaerobic respiration. Therefore, it is often used in the study of environmental remediation. Under anaerobic condition, electrons, produced by catabolism of carbon source in cytoplasm of S. oneidensis MR-1, are transferred through multiple electron transfer systems which were composed by 42 types of c-type cytochromes or reductases located in inner membrane to outer membrane, and finally to electron acceptors out of the cells. In this review, types of electron transfer mechanism for anaerobic respiration are introduced to facilitate a deeper understanding to the mechanism of pollutant biodegradation and nanomaterial synthesis. Thus, this review is beneficial to providing an adequate theoretical basis for the application of this strain in bioremediation

Abstract:In this article, I share my teaching experiences, and explore ideas on teaching Microbial Genetics in the post-genome era. To increase student interest in this course, I propose the use of story-telling and visualization as two teaching strategies. In addition, to help students better understand post-genomic approaches, I suggest integration of traditional genetics and related post-genomics (examples of integration include traditional forward genetics and fast forward genetics, phenotypic analysis of single-gene deletion and genome-wide deletion mutant analysis, and traditional genetic interactions and genome-wide genetic interactions). Also, introducing online bioinformatics resources extends the classroom teaching, and improves teaching quality.