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Abstract:[Objective] The purpose of this study was to characterize the isolate MF1006, and to determine the taxonomy and pathogenicity of the spiroplasma obtained from diseased hon-eybees in China. [Methods] The spiroplasma morphology was examined by dark-field micro-scope and transmission electron microscope. The biological characteristic of the spiroplasma was investigated by using conventional culture-dependent method and molecular biology and serological methods. We determined the spiroplasma pathogenicity through feeding tests. [Results] MF1006 exhibited typical properties of spiral morphology and mobility. It was able to pass through membrane filters with pores size of 220 nm. This isolate was very different from the previous reported domestic isolate Spiroplasma melliferum CH-1 which caused hon-eybee spiroplasmosis seriously. S. melliferum CH-1 antiserum could not inhibit MF1006. Phylogenetic analysis based on the 16S rDNA and ITS revealed that MF1006 was close to S. apis which was found in France and associated with a lethal infection (“May disease”) for honeybee. In addition, MF1006 expressed strong pathogenicity to A. mellifera. [Conclusion] MF1006 was the second pathogenic spiroplasma to honeybees in China.

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Abstract:[Objective] The purpose was to clone and express endo-1,4-β-glucanase gene of Humicola insolens in Pichia pastoris expression system. [Methods] An endo-1,4-β-glucanaseⅡ(egⅡ) cDNA gene was isolated from the fungus Humicola insolens NC3 by RT-PCR. Subsequently, we cloned the egⅡgene into expression vector pPIC9K, and then transformed into Pichia pastoris GS115. [Results] SDS-PAGE and CMC enzyme activity analysis demonstrated that recombinant EGⅡ protein was successfully expressed after induction in shake flasks. The endo-1,4-β-glucanase exhibited maximum activity at 70 °C and pH 6.5, and was stable between pH 6.0 and 7.0 and below 65 °C. [Conclusion] P. pastoris expression system is an efficient way of production of endo-1,4-β-glucanase. The recombinant endo-1,4-β-glucanase could be a candidate for industrial applications.

Abstract:[Objective] The aim of the present study was to isolate and screen potential nitrite removal bacteria, and examined the nitrite removal conditions. [Methods] Nitrite removal bacteria were first isolated from the aquaculture sediment, and the potential nitrite removal isolate was further screened through the nitrite removal rate comparison, identified using API ID32GN bacterial identification system and 16S rDNA sequence analysis, and its nitrite removal conditions were studied using single factor method. [Results] A potential nitrite removal bacterium AQ-3 was isolated and screened from the aquaculture sediment, its removal rate to 50 mg/L nitrite was 99.47%. Strain AQ-3 was identified as Acinetobacter baumannii (GenBank accession number: JF751054.1). Its 16S rDNA sequence had homology of 99%?100% with those of Acinetobacter sp. strains submitted to GenBank, and showed the most close relative to Acinetobacter baumannii strain KF714 (GenBank accession number: AB109775). The optimum initial pH range for the nitrite removal of strain AQ-3 was from 7 to 9, and the optimum carbon sources for its nitrite removal were sodium acetate and sodium succinate. In addition, the nitrite removal rates of strain AQ-3 were gradually rising with the increase of its initial bacterial concentrations, and the nitrite removal rates were gradually reduced with the increase of nitrite content. [Conclusion] The present study enriched the resources of nitrite removal bacteria and provide scientific basis to the practical use of strain AQ-3 in aquaculture.

Abstract:[Objective] In order to study on the characteristics of denitrifying and phosphorus removal bacteria. [Methods] Many strains with denitrification and phosphorus removal characteristics under aerobic condition were isolated by screening of microorganism and methods of biological characteristics from the water and sediment samples of shrimp culture ponds. [Results] Among them strain LY-1 could remove nitrite nitrogen from 10 mg/L to 0.04 mg/L, and the PO43?-P from 10 mg/L to 0.05 mg/L in 18 h, respectively; and approximately to 100% of denitrifying and phosphorus removal rate were reached at the DO concentration of 5.0?5.9 mg/L. Furthermore, in comparison with a denitrifying and phosphorus removal bacterium, Ba-cillus subtilis selected as the positive control and Escherichia coli as the negative control, strain LY-1 was tested under different pH, temperature, salty, PO43?-P and nitrite concentra-tion, its denitrifying removal rate was reached approximately 99%, and phosphorus removal rate reached 86% at the pH of 5?9; the denitrifying and phosphorus removal rate of strain YX-6 reached proximately 100% at 30 °C; The rate almost reached 99% when salty ranged between 5 and 15, phosphorus concentration was 10 mg/L and nitrite nitrogen concentration was 20 mg/L. [Conclusion] The results showed that the denitrifying and phosphorus removal of LY-1 was significantly higher than that of the two controls (P<0.05). According to the mor-phological, physiological and biochemical properties, and the analysis of its 16S rRNA gene sequence, strain LY-1 was identified as Bacillus cereus primarily.

Abstract:[Objective] Competitive nodulation ability of 12 rhizobial strains associated with Medicago polymorpha is tested by using BOX-PCR Molecular fingerprint; effective rhizobial strains were finally confirmed using the nodule occupancy rate combined with plant growth. [Methods] The nodule occupancy rate calculated by comparing BOX-PCR Molecular finger-prints of inoculated strains with those part of re-isolated strains. [Results] It showed that nod-ule occupancy of strain SWF67523 from Yingjiang district was of the highest occupation nodulation rate, reach 93.33%, showing strong ability of competitive nodulation, and it in-creased the dry-weight of their host-plants for 100% by plant test; while the other strain SWF67409 was of lower nodule occupancy rate, but with highly nodulation rate and increased the dry-weight of their host-plants for 106.5%. Furthermore, strain SWF67394 obviously in-creased the dry-weight of their host-plants and with high nodulation rate and lower nodule occupancy rate, these two strains may had effect on influencing activities of the native rhizobia and other microorganisms in soil. [Conclusion] We have introduced the competitive nodula-tion ability in screening effective rhizobial strains, and obtained a strain SWF67523 that with highly competitive nodulation ability and highly ability of plant growth promoting, and plant growth promoting strains of SWF67409 and SWF67394, this will provide precarious strains resources for producing rhizobial inoculants.

Abstract:[Objective] The definite relation was found between polymorphism, pathogenicity differentiation of Phytophthora capsici Leonian and regionality in Liaoning Province. [Methods] Using Sequence related Amplified Polymorphism (SRAP) technology, the 25 strains of Phytophthora capsici Leonian in Liaoning were amplified in PCR method and evaluated by clustering analyses of NTSYS-PC. The pathogenic differentiation was researched by root-drenching method, and it was examined with hierarchical cluster combination by SPSS 11.5. [Results] The results showed that the DNA of 25 strains were amplified by 27 primer pairs. A total of 578 bands were identified, of which 548 bands were polymorphic. The percentages of polymorphic fragments of each primer pairs were ranged from 84% to 100%. There were abundant polymorphism clustering analysis showed that the genetic similarity among the 25 strains of Phytophthora capsici Leonian were very high. The similarity coefficients varied from 0.56 to 0.91. They were divided into four groups at 0.68 for similarity coefficient, significant correlations were not obvious among the sources of these strains, and there were not regional characteristics. 80% strains were intermediate pathogenicity. [Conclusion] The tested strains were not significantly regional characteristics, and there was no regularity of pathogenicity differentiation from different areas.

Abstract:[Objective] Bioinformatics analysis revealed that the 89K pathogenicity island (PAI) of highly pathogenic Streptococcus suis serotype 2 (SS2) in China encodes a putative type II toxin-antitoxin (TA) system named SezAT, which is homologous to the epsilon-zeta system from S. pyogenes. SezAT is presumed to be requisite for the stability of the 89K PAI in SS2. To confirm the SezAT system is a functional TA system. [Methods] The sequence characteristics of SezAT were subjected to further bioinformatics analysis. RT-PCR was performed to analyze the transcriptions of the sezAT locus and the flanking genes. The SezT toxin and SezA antitoxin proteins were selectively overexpressed in Escherichia coli. Deletion mutagenesis was carried out to obtain a SezAT-deficient mutant. [Results] Bioinformatics analysis and RT-PCR results suggest that sezAT are in the same operon. Overexpression of SezT led to severe growth inhibition of the host bacteria, while this toxicity was counteracted by the expression of SezA. Finally, the toxin-encoding gene sezT was successfully knockout by allelic replacement. [Conclusion] All of these results suggest that SezAT is an activated toxin-antitoxin (TA) system. Moreover, our results provide foundations for investigating the potential stabilized effect of SezAT in the 89K PAI, and screening an 89K-negative mutant to better understand the pathopoiesis of 89 K in highly pathogenic SS2.

Abstract:[Objective] In order to improve production of α-glucosidase inhibitors from Streptomyces gobitricini PW409, response surface methodology was used to optimize the fermentation medium. [Methods] A Plackett-Burman design was used to evaluate the influence of seven factors firstly. Then, the path of steepest ascent and the Box-Behnken design were adopted for further optimization, the optimum concentration levels and the relationships among these fac-tors was found out by quadratic regression model equation with Design-Expert statistic methods. The concentrations of α-glucosidase inhibitors in the fermentation broth were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). [Results] The concentration of soluble starch, KNO3 and K2HPO4 played important roles in influencing the content of inhibitors. The optimal fermentation medium is: soluble starch 9.01 g/L, KNO3 11 g/L, K2HPO4 0.32 g/L, MgSO4?7H2O 0.50 g/L, FeSO4?7H2O 0.01 g/L, pH 7.5. [Conclusion] Under such conditions, the half maximal inhibitory concentration (IC50) of fermentation broth to maltase glucoamylase was 22 mg/L, inhibitory activity increased nearly 10 times. The concentration of 1-deoxynojirimycin and miglitol were increased to 7.84 mg/L and 0.94 mg/L, which was 668 and 10 times higher than before, respectively.

Abstract:[Objective] To obtain a fungus capable of reducing N, N-dimethyl-3-keto-3- (2-thienyl)-1-propanamine (DKTP) to (S)-DHTP, a key precursor to synthesize Duloxetine. [Methods] Routine and an improved biotransformation method were used to screen fungus from soil, and the type of the fungus was identified by analysis of morphological characters and DNA sequence of 26S rDNA. [Results] A fungus was obtained which was capable of asymmetrically reduce DKTP to (S)-DHTP with high yield (more than 90%) and high enanti-ometric excess (more than 99%), and the improved method was more convenient and efficient. The identification showed that the fungus belong to the Genus of Rhodotorula Harrison, and was named as Rhodotorula sp. 507. [Conclusion] The screened fungus could efficiently and asymmetrically reduce DKTP to (S)-DHTP, which makes it possible to get enough precursor for the synthesis of Duloxetine easily and economically.

Abstract:[Objective] To construct the polyphosphate kinase 1 gene deletion mutant of Escherichia coli (E. coli) K1 strain E44 and explore the role of ppk1 in the pathopoiesis of meningitis E. coli K1. [Methods] The ppk1 gene was knocked out from the genome of E. coli K1 strain E44 by using suicide vetor pCVD442 and homologous recombination, and the mutant was named Δppk1. Then the survival ablities of E44 and Δppk1 in poor nutrition condition and oxidative stress were examined. The ability of the mutant adhering to human brain microvascular endothelial cells (HBMEC) was also compared with that of the wild type. The cytogenetic toxic effects induced by Δppk1 and E44 were tested by using the lactic dehydrogenase testing kit. [Results] The ppk1 gene of the mutant had been knocked out and was confirmed by PCR and DNA sequencing. The ppk1 deletion mutant showed to be defective in adhesion ability to HBMEC and survival abilities under poor nutrition condition and oxidative stress. The damaging effects of HBMEC induced by Δppk1 were significantly less than that induced by E44. [Conclusion] The ppk1 gene plays an important role in E. coli K1 surviving in low nutrition and oxidative stress condition, adhering to HBMEC and inducing cell injury.

Abstract:[Objective] To validate the immune protective efficacy of Chlamydia secretion protein Pgp3 and to analyze the potential immune mechanisms related to this protection. [Methods] The prokaryotic recombinant protein Pgp3 was purified and evaluated for its pro-tective efficacy in a genital tract infected mouse model. Groups of BALB/c mice were immu-nized intranasally or intramuscularly with adjuvants plus Pgp3 protein or PBS and GST con-trol. Humoral and cellular immune responses were evaluated. After Chlamydia muridarum in-travaginal challenge, the chlamydia shedding from the lower genital tract and the chlamy-dia-induced upper genital tract gross pathology and histopathological characterization were also detected. [Results] Adjuvant plus Pgp3 immunization can induce high level of Pgp3 spe-cific antibody as well as IFN-γ, IL-17 and IL-5 cytokine production. More importantly, intra-nasal immunization compare with intramuscular route induced more Th1-dominant immunity that significantly reduced the shedding of live organisms from the lower genital tract and at-tenuated inflammatory pathologies in the oviduct tissues. [Conculsion] These observations have demonstrated that secretion protein Pgp3 intranasal immunization can induce protective immunity against chlamydial infection and pathology in mice.

Abstract:It is well accepted that microbial population from the environment is complex and contains high abundance of gene resources. Researches on uncultured microbes can be done more profoundly with the advent of molecular tools. Metatranscriptomics, a recently developed technology, is one of the important tools that can be used for understanding biologically active functional genes and their pathways. In this mini-review, we described the concept of metatranscriptomics and its advantages over metagenomic approaches, with emphasis on the research methods for metatranscriptomics and its applications.

Abstract:Multidrug transporter Bmr is one of the main drug resistance efflux proteins in Bacillus subtilis. It’s encoded by bmr gene which is located in the genome, and it mediates the resistance to a wide range of drugs such as antibiotics and antimicrobials, etc. The expression of bmr gene is regulated by BmrR and MtaN, both of which are transcriptional regulator from the MerR family. This paper reviewed the study of structure, physiological function and the action mechanism of Bmr and the regulatory protein BmrR and MtaN in recent years.

Abstract:Pretreatment of lignocellulosic materials for ethanol production generates inhibitory compounds that interfere with microbial growth and subsequent fermentation. To facilitate fermentation process, detoxification techniques have been commonly applied to remediate these inhibitory compounds. However, these additional steps result in sugar loss and an extra cost in practice. To meet desired overall yields during ethanol production, it is of great practi-cal importance to improve the inhibitor-tolerance of Saccharomyces cerevisiae. In the last decade, significant progress has been made in understanding the mechanisms of yeast stress tolerance. This article reviewed the molecular mechanisms of Sacchromyces cerevisiae inhibi-tor tolerance focusing on its enhanced expression and pathway analysis of some key genes. We also discussed the strategies for improving the fermentation performance of yeast.

Abstract:[Objective] The study aimed to develop an efficient and sensitive high-throughput method to obtain alpha-amino acid ester hydrolase (AEH) with improved activity or thermo-stability. [Methods] Standard curve was made based on the fact that hydrolysis of cefaclor in alkaline buffer yields a derivate which has specific absorbance at 340 nm. Whole cell-based ultra-violet spectrophotometric method was applied to screen the cefaclor synthesis activity of AEH variants at a high-throughput scale. [Results] Beer’s Law is obeyed in the range of (0.1?0.6)×10?3 mol/L cefaclor. The average recovery is 99.8%?101.3%. 2 300 Clones obtained by one round of site-directed saturated mutagenesis were screened by this method. Three variants with more than 1.4-fold kcat and 4 variants with T50 5 °C more than wild type were ob-tained. [Conclusion] The screening method was precise and reliable. The screen capacity can be up to 2 000 samples per day, which was in the scale of high-throughput screening.