Abstract:[Background] Although the coal mine wastewater treatment by constructed wetland has succeeded remarkably, few studies can be found on the structure and function of microbial communities in constructed wetlands sediment. [Objective] The purpose of this study is to clarify the effect of constructed wetland on the coal mining wastewater treatment and the structure and function of microbial community. [Methods] The water quality at different sampling sites of constructed wetland in Zuoyun, Shanxi was monitored. Based on high throughput sequencing technology, V3?V4 region of the bacterial 16S rRNA gene in constructed wetlands sediment were sequenced. Then the composition of bacterial communities in sediment were analyzed. [Results] The quality of wastewater from coal mine was effectively improved after flowing through the constructed wetland. Removal efficiencies of biochemical oxygen demand (BOD5), chemical oxygen demand (CODCr), total nitrogen (TN) and total phosphorus (TP) in constructed wetlands were 76.2%, 93.4%, 73.4%, 99.3%, respectively. Illumina sequencing of 16S rRNA amplicons revealed that a total of 2 832 operational taxonomic units (OTUs) were detected. Sequence alignment results showed that a total of 51 bacterial phyla, 150 classes, and 753 genera were found. Among the 4 sampling sites, the dominant bacteria phyla were Proteobacteria (50.0%?64.7%) and Bacteroidetes (15.8%?21.2%). The sampling sites of M1, M2, and M3 had the highest abundance of β-Proteobacteria and γ-Proteobacteria, which were rich in ammonia nitrogen oxidation bacteria. The sampling site of M4 had the greatest number of bacterial OTUs with the highest diversity. There were significant positive correlations between the relative abundance of Azoarcus and contents of the TN, while significant negative correlations between the relative abundance of Sulfurimonas, Sulfuricurvum and the contents of NH4+-N. Moreover, the relative abundance of Novosphingobium was also positively correlated with the contents of the TP. [Conclusion] The effective treatment of coal mine wastewater is achieved after flowing through constructed wetland, and bacterial diversity plays a decisive role in the ecological function of constructed wetland.

Abstract:[Background] The Huixian wetland, located in the transition zone between the karst peak-cluster plain and karst depression area of Guilin city, is a typical karst wetland with a calcium-rich, alkalescent underground water supply. The sediment of the wetland is mostly free of oxygen, making it an ideal place to study novel species of anaerobic microbes. Hydrogenotrophic and acetoclastic methanogens play significant roles in the degradation of organic matter in the anaerobic environment; however, the physiology and ecology of microbial methanogenesis within the Huixian karst wetland is poorly understood. [Objective] To reveal the variation of archaeal communities, the functional groups of methanogens present, and the potential interactions among archaea from the sediment of the Huixian wetland under hydrogenotrophic and acetoclastic methanogenic conditions. [Methods] Sediment samples were incubated in anaerobic basal medium supplemented with hydrogen or acetate as the sole energy source. During incubation, the methane concentration in the headspace of the incubator was monitored. After 21 days of incubation, 3 libraries of nearly complete archaeal 16S rRNA gene sequences including 146 sequences and 3 mcrA (methyl-coenzyme reductase gene) libraries including 136 sequences from different samples were constructed. The dynamics of the archaeal communities in response to different conditions were studied through phylogenetic analysis. [Results] Methane was detected in the headspace of both H2- and acetate-enriched samples. The mcrA libraries were dominated by Methanosarcinales archaea consisting of four major phylogenetic branches: Zoige cluster I (ZC-I), anaerobic methanotrophic archaea of ANME-2d (AD), and the KT-I and KT-II subgroups. The branches of KT-I and KT-II were comparatively independent in the phylogenetic tree, with no previously reported sequences included, and these branches might represent novel subgroups of Methanosarcinales archaea. The composition of ZC-I, AD and KT-II increased by 45%–155% after incubation under methanogenetic conditions. Bathyarchaeota archaea accounted for 88%–100% of the 16S rRNA gene libraries. Most of the Bathyarchaeota came from the MCG-11 subgroup, which increased by 17% after incubation under acetic methanogenic conditions. [Conclusion] The sediment of the Huixian wetland contains novel archaeal sequences. The dominant hydrogenotrophic and acetoclastic methanogens within the wetland sediment are archaea of the Methanosarcinales order. Bathyarchaeota may play important roles in both in situ karst environments and methanogenic conditions.

Abstract:[Background] Jinsha earthen site is considered as a large ritual site in the Shang and Zhou dynasties 3 200 years ago, which play an important role in ancient culture and history. But now it has exhibited different degrees of degradation under the influence of physical, chemical and biological factors. The effects of physical and chemical factors have been reported, while very little is known about the influence of biological factors on earthen site. [Objective] To study the microbial diversity and metabolic characteristics in Jinsha earthen site soil, it is important to provide scientific evidences for the conservation of Jinsha earthen site. [Methods] Four types of representative samples from Jinsha earthen site that have undergone different degree of degradation were collected to analyze the microbial diversity and metabolic function by Biolog plate methods and denaturing gradient gel electrophoresis (PCR-DGGE). The degree of degradation of samples is followed by J4>J3>J2>J1. [Results] Biolog analysis result showed that the function of soil microbial diversity was significantly varied in different samples and the order of microbial metabolic activity was followed by J2>J3>J4>J1, implying that the microbial metabolic activity of samples showed an increasing trend with the degradation of soil. PCA analysis of Biolog indicated the utilized types of carbon sources by soil microbes in Jinsha earthen site was significantly varied. The microorganisms in sample J2 could utilize most types of substrate carbon sources in plate compared to that in other samples, and the preferred carbon sources of sample J2 was obviously different from that of other samples. The bacterial diversity and structure of different soil exhibited significant difference by DGGE analysis. The order of microbial community diversity was followed by J2>J4>J3>J1 both in DNA and RNA level and the PCA result of DGGE revealed that the total and active bacterial community showed high consistency except sample J1, which indicated the microbial diversity increased with the degradation of soil. Based on the sequence analysis of DGGE, the major community of bacteria in soil samples were belonged to Actinobacteria, Acidobacteria, Proteobacteria and Deinococcus-Thermus. Rubrobacter and Brevundimonas were detected in all samples, including DNA and RNA level. [Conclusion] It is first time that microbial and functional diversity of soil from Jinsha earthen site were analyzed in this study. The results exhibited the microbial and functional diversity increased with the degradation of soil. The genera Rubrobacter, Tellurimicrobium and Brachybacterium that were detected only in degraded soil samples or had high expression activity may be involved in the soil degradation process. The results could provide theoretical basis for the scientific conservation of Jinsha earthen site.

Abstract:[Background] Escherichia coli BL21(DE3) is a commonly used host for genetic engineering. It synthesizes 5-aminolevulinic (ALA) through C5 pathway. ALA is an important precursor, while its secretion on the heme synthesis is unclear. [Objective] To elucidate the role of RhtA in the export of ALA in the heme synthesis pathway. [Methods] rhtA was knocked out by Red homologous recombination, and plasmid pEA was constructed to overexpress hemA, a key enzyme gene in the heme biosynthesis pathway. The contents of heme and its precursors as well as the transcription levels of 10 key genes in the heme synthesis pathway were analyzed. [Results] The deletion of rhtA had no significant effect on cell growth. Compared with parental strain BL21(DE3), the extracellular content of ALA decreased by 23% while the content of heme increased by 12% in the knockout strain BL21(DE3)ΔrhtA. The contents of uroporphyrin III (UIII), coproporphyrin III (CIII) and protoporphyrin IX (PPIX) increased by 25%, 15% and 18%, respectively in BL21(DE3)ΔrhtA. Compared with the strain BL21(DE3)/pEA in which hemA was overexpressed, the content of extracellular ALA reduced by 16% and the content of heme increased by 24% in the strain BL21(DE3)ΔrhtA/pEA. And the contents of UIII and CIII increased by 55% and 64%, respectively. The content of PPIX increased significantly, about 4.7 times in BL21(DE3)ΔrhtA/pEA compared with that in BL21(DE3)/pEA. The results of real-time quantitative PCR showed that after the deletion of rhtA, the transcription level of hemC was down-regulated, while those of the other 9 genes were up-regulated to various degrees. [Conclusion] The rhtA knockout reduces the export of ALA and leads to an increase in intracellular heme production.

Abstract:[Background] In order to develop new microbial resources in the ocean, our laboratory constructed a deep sea metagenomic library by adopting the culture-independent metagenomic technology, and carried out follow-up studies on the important genes. [Objective] To identify and highly express the methionine γ-lyase gene in Escherichia coli from the DNA library of deep-sea sediments. [Methods] The gene mgl was overexpressed by pET-28a(+) system in E. coli BL21(DE3), which was induced by isopropyl β-D-1-thiogalactopyranoside, and the expression conditions were optimized to obtain the high expression of methionine-lyase (rMGL). The recombinant protein was purified by affinity chromatography and the enzyme activity was studied. [Results] The product rMGL was consistent with the predicted 46 kD, with high L-methionine lyase activity. rMGL could use L-methionine and DL-homocysteine as substrate, but had little activity on L-cysteine and L-cystine. Its relative activity on DL-homocysteine was 1.4 times of that on L-methionine. [conclusion] mgl gene from the deep sea metagenomic library can efficiently express rMGL using pET-28a(+)/BL21(DE3).

Abstract:[Background] Ganoderma lucidum polysaccharide (GLP), as a macromolecular substance with various biological activities, has attracted a lot of attention in recent years. Some researchers have improved the yield of GLP through optimization of fermentation regulation or strain improvement for G. lucidum. However, it is a fact that the biosynthesis pathway of GLP is still unclear completely, and the understanding about characteristics of key enzymes involved in GLP synthesis are insufficient. Thus, there is a bottleneck on substantially improving the yield of GLP. [Objective] Through heterologous expression of the genes of phosphoglucomutase (PGM), UDP-glucose pyrophosphorylase (UGPG), and phosphomannose isomerase (PMI) cloned from G. lucidum in E. coli, decent amounts of the purified enzymes were obtained. We characterized their enzymatic properties and compared them with other sources, to provide insights into the characteristic information of them, and build an efficient strategy for the synthetic of GLP on fermentation. [Methods] Genes of gl-pgm, gl-ugpg and gl-pmi were cloned from G. lucidum strain CGMCC 5.26 and expressed in E. coli, the recombinases were purified with Co-NTA resin respectively. Then, the enzymatic properties of the purified enzymes were studied. [Results] GLpgm, GLugpg and GLpmi, were successfully expressed in E. coli. The optimum reaction pH of GLpgm was 8.5, and it was 7.5 for GLugpg and GLpmi. The optimum reaction temperature of GLpgm, GLugpg and GLpmi were 35, 40 and 30 °C, respectively. For them, 1 mmol/L of Ag+ and Cu2+ had strong inhibitory effects, while Mn2+ and Mg2+ had activation effects on GLpgm and GLpmi, especially the use of Mn2+ could increase the GLpgm activity by 2.7 times. The kcat/Km values of GLpgm, GLugpg and GLpmi were 196.08, 818.60 and 1 105.22 mmol/(L·s), respectively. [Conclusion] GLpgm, GLugpg and GLpmi were similar to the sources of plant and other fungi in terms of optimum pH, temperature and metal ions action, while the catalytic efficiency was relatively higher than them. Their properties could provide more informantion for the regulation of GLP based on it’s pathway.

Abstract:[Background] Banana Fusarium wilt is a devastating disease caused by the soil-borne hyphomycete, Fusarium oxysporum f. sp. cubense (Foc). Due to the low production limitation of resistant cultivars and attempts to control Fusarium wilt with chemical fungicides have proved uneconomic and are environmentally unfriendly, using novel strategies such as biological control is attractive alternatives to conventional control methods. [Objective] In order to select dark septate endophytes strains that showed significantly antagonistic effect on the Foc growth and to enrich the microbial resources for biocontrol. [Methods] Five dark septate endophytes strains were selected for evaluation of banana growth promotion ability and control efficacy against the Fusarium wilt through culture dish and pot culture method, and the dominant strain LS1 was identified based on morphological characteristics and ITS sequence analysis. [Results] All the five dark septate endophytes strains have the ability to carry out banana growth promoting activities after the inoculation, but the strain LS1 function most notably, which indicated by the seedling fresh weight and dry weight were increased by 47.36% and 42.40%, separately. The disease control efficacies of LS1 against the Fusarium wilt was found up to 86.19% in the culture dish and 63.19% in the pot culture method, which were significantly higher than other dark septate endophytes strains. Morphological observation and ITS sequence alignment analysis results showed that the LS1 strain belonged to Cladosporium chlorocephalum. [Conclusion] These results indicated that the strain LS1 promised to provide a new biological control method for banana Fusarium wilt disease.

Abstract:[Background] Streptomyces venezuelae Snea253 is an actinomycete with nematicidal activity to plant-parasitic nematode. Bioinformatics analysis showed that γ-aminobutyrate transaminase gene (gabT) is one of the important genes involved in carbon metabolism in Snea253. [Objective] The purpose of this research is to clarify that the gabT gene affects the activity of the strain by regulating the γ-aminobutyric acid metabolic pathway of Snea253. [Methods] First, the mutant strain (Snea253-R) obtained by UV mutation was used to overexpress the gabT gene, and Meloidogyne incognita J2 was used as the target nematode. γ-aminobutyric acid and the metabolite succinic acid were detected by enzyme-linked immunoassay (ELISA) and high performance liquid chromatography (HPLC), and the nematicidal activity was also detected. Meanwhile, the gabT gene expression level, product content and nematicidal activity of wild-type strain with eight carbon sources culture were detected. [Results] The expression of gabT gene in overexpression strain R-pIB139 was up-regulated, γ-aminobutyric acid content was decreased, the content of succinic acid was increased, and the nematicidal activity was increased by 39% in comparison with the control. In eight carbon sources cultures, the carbon source medium with the higher relative expression of gabT gene in wild plants was soluble starch and corn starch. Under this condition, γ-aminobutyric acid content in fermentation broth was lower, the other products of metabolism were increased, and the nematicidal activity was higher. [Conclusion] By changing the expression of gabT gene, we confirmed that γ-aminobutyric acid shunt plays an important role in regulating the metabolism of Snea253 to enhance nematicidal activity.

Abstract:[Background] Virus infects sweet potato plants, and causes serious economic losses frequently in Fujian province. There may exist more than one species of sweet potato virus. [Objective] To determine the sweet potato virus species and prevalence in Fujian, and analyze the genetic diversity of the main viruses. [Methods] Viruses were isolated from samples with viral diseases symptoms from different sweet potato production areas in Fujian province, 14 pair primers of viruses infecting sweet potato were used to detect by PCR or RT-PCR. The PCR amplicons of SPFMV (sweet potato feathery mottle virus), SPCSV (sweet potato chlorotic stunt virus) and SPLCV (sweet potato leaf curl virus) from selected samples were cloned and sequenced. The alignments and phylogenetic analyses were performed with the MEGA 6.0 software package. [Results] The results showed that 12 virus species were detected infecting sweet potato in Fujian, of which 9 were RNA viruses, including sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato virus G (SPVG), sweet potato virus C (SPVC), sweet potato virus 2 (SPV2), sweet potato chlorotic fleck virus (SPCFV), sweet potato latent virus (SPLV), sweet potato mild speaking virus (SPMSV), cucumber mosaic virus (CMV); and three DNA viruses including sweet potato leaf curl virus (SPLCV), sweet potato symptomless virus 1 (SPSMV-1), sweet potato badnavirus B (SPBV-B). Among 179 sweet potato samples, SPFMV was detected in 90 samples (50.28%), SPCSV was detected in 75 samples (41.90%), SPVG in 64 samples (35.75%), SPVLCV in 44 samples (24.58%), and CMV just in five sample (2.79%), however, no samples infection was detected for SPV-2 and SPMMV. Mixed infections of 2–6 viruses were common (85.61%) in Fujian. The analysis of phylogenetic relationships showed that SPFMV isolates mainly belong to strains of EA, O and RC, SPCSV isolates mainly belong to strains of WA, and SPLCV isolates were divided into two groups. [Conclusion] There were many sweet potato viruses with complex infection models and genetic diversity were proved in Fujian province.

Abstract:[Background] Siderophores is considered as a new bioactive substance with development and utilization value, and has been gradually applied to the control of plant pathogens. [Objective] To clarify the optimal fermentation conditions for the production of siderophores by rhizosphere growth-promoting bacteria Rahnella aquatilis JZ-GX1 and further explore the potential role of siderophores in controlling plant root diseases. [Methods] Several fermentation factors affecting the secretion of siderophores were studied by CAS fermentation analysis, and the antagonistic effect of siderophores on two forest pathogenic fungi was determined by mycelial growth inhibition rate method. [Results] The results showed that KMB-based medium, initial pH 8.0, liquid volume 25 mL/50 mL, inoculated with 1%, and cultured at 28 °C for 36 h, can obtain higher yield of siderophores; it can produce carboxylate and hydroxamic acid type siderophores; under optimal conditions, the inhibition rate of the fermentation stock solution against Phytophthora cinnamomi and Rhizoctonia solaniwas were 100%. [Conclusion] Rahnella aquatilis JZ-GX1 has a good potential for controlling root diseases of forest trees in alkaline soils.

Abstract:[Background] Fusarium oxysporum f. sp. cubense race 4 (Foc4) has been a life-threatening disease to banana production. Present studies have shown that soil pH is negatively correlated to the onset of banana wilt disease. However, most studies of influence of the pH on growth of Foc4 of banana were done under the condition of the pH being adjusted with strong acid and alkali, without tested the terminal pH of the test medium. In fact, the investigation of the pH on Foc4 is not systematic and the results of the study are not practice to guide farmers to remedy the Foc4 disease by modifying the soil pH. [Objective] to study the effect of pH on growth, sporulation and spore germination of Foc4 as well as the side effect of growth, sporulation and spore germination of Foc4 on the environmental pH throughly. [Methods] Laboratory incubations with 9 pH treatments from pH 3.0 to pH 11.0 were applied in the study. [Results] The most suitable condition for growth, sporulation and spore germination of the Foc4 is weak acid to neutral environment, being pH 5.0 to 7.0. The average germination rate of spores in weakly alkaline environment (pH 8.0 and pH 9.0) was decreased by 73.1% compared to that in weakly acidic environment (pH 5.0 and pH 6.0). Compared with the acid treatment under pH 6.0, the sporulation rate under pH 8.0 and pH 9.0 decreased by 52.3% and 68.1%, respectively. [Conclusion] The growth and germination of Foc4 is able to secrete acid. However, terminal pH of the liquid medium has been stable when pH buffer system is used in the medium to culture the Foc4, except for treatment of pH 9.0 and pH 10.0. The terminal pH of the treatment of pH 9.0 and pH 10.0 only decreased by 0.34 and 0.27 units respectively. The results indicated that the reliable results of the effect of pH on the Foc4 growth and germination should be under the pH buffer system liquid medium. Within the pH range where the crop can grow (pH 5.0?9.0), alkaline and slightly alkaline conditions (pH 8.0?9.0) are capable to inhibit the growth, sporulation and spore germination of Foc4 significantly.

Abstract:[Background] The traditional koumiss is naturally fermented by fresh mare’s milk, and the quality of koumiss is greatly affected by the bacteria in fresh mare’s milk. However, the microbiota composition in fresh mare’s milk was rarely investigated. [Objective] In order to explore the microbial variation from fresh mare’s milk to koumiss, microbial diversity of fresh mare’s milk was investigated, to provide a reference for the industrialized production of koumiss. [Methods] Six fresh mare’s milk samples were collected from herdsmen families of Xilinhot in Inner Mongolia, China. The bacterial full-length 16S rRNA gene sequences in all samples were sequenced by PacBio SMRT sequencing technology to analyze the bacterial composition and diversity. Meanwhile, the structure and diversity of bacteria in fresh mare’s milk and koumiss were compared. [Results] The results showed a large number of lactic acid bacteria was detected in the fresh mare’s milk such as Lactococcus lactis (12.43%), Lactococcus garvieae (3.53%) and Leuconostoc mesenteroides (1.77%). In addition, some opportunistic pathogens were also detected such as Cronobacter sakazakii. Compared with koumiss, the bacterial diversity in fresh mare’s milk were significantly higher, and Lactococcus lactis and Lactobacillus helveticus were found both in fresh mare’s milk and koumiss. In fresh mare’s milk, Leuconostoc mesenteroides correlated with Lactococcus lactis and Lactococcus garvieae positive, negative related with Aeromonas hydrophila, which implied lactic acid bacteria may suppress pathogen bacteria during the fermentation. [Conclusion] During the fermentation of koumiss, the growth of lactic acid bacteria could inhibit pathogen bacteria, decrease the bacterial diversity and improve the quality of koumiss.

Abstract:[Background] Acetic acid-tolerant lactic acid bacteria are important for lactic acid and its derivatives production during acetic acid fermentation in traditional cereal vinegar brewing process. [Objective] To screen acetic acid-tolerant lactic acid bacteria from vinegar Pei of Zhenjiang aromatic vinegar, and evaluate its fermentation performance. [Methods] Selective MRS medium containing 4% (volume ratio) acetic acid was used to isolate acetic acid-tolerant lactic acid bacteria. The isolated strain was identified by 16S rRNA gene sequencing, whole genome sequencing and morphological, physiological and biochemical analyses. Lactic acid-producing ability of the isolated stain was evaluated under different culture conditions. [Results] A lactic acid bacterial strain JN500903 that resisted 6% acetic acid was screened and identified as Lactobacillus sp. The optimum conditions for lactic acid production were: anaerobic static culture, inoculum of 5%, acetic acid concentration of 5%, glucose concentration of 40 g/L, fermentation temperature at 37 °C and fermentation period of 10 d, the lactic acid production was 16.1 g/L under these conditions. [Conclusion] Strain JN500903 can produce lactic acid under the 6% acetic acid stress, suggesting its potential for industrial application.

Abstract:[Background] White pellicle was always visible on the surface of soybean paste, which seriously decreasing consumers’ desirability and thus leading to an enormous waste of the product. [Objective] This study was designed to avoid the emergence of white pellicle in soybean paste by developing novel, safe and targeted control strategies. [Methods] First, the microstructure of white pellicle was visualized by light microscope. Second, microorganisms were selected from white pellicle using the YPD medium containing 18% NaCl. Then, species of these selected microorganisms were identified according to ITS (Internal transcribed spacer) sequence analyses and carbon source utilization experiments. Finally, the molecular mechanisms of white-pellicle-formation were analyzed. [Results] The white pellicle was visualized as repeated, round and movable body by light microscope and microorganisms which were selected from the white pellicle were identified as velum-forming Zygosaccharomyces rouxii. On the one hand, when oxygen was restricted, the transcription of ACS2 (a lipid biosynthetic gene) and FLO11 could be inhibited, and the reduction of lipid synthesis rate may have a negative effect on the propagation of yeast, and thus the production of the white pellicle was inhibited. On the other hand, the addition of 2% bioethanol can significantly enhance the transcription of protein transport-coding gene BTN2, and thus prevented the formation of white pellicle, eventually. [Conclusion] The study revealed the cause of white pellicle in soybean paste and proposed targeted strategies for the control of white pellicle without reducing the flavor and taste, which provided a new idea for the storage and consumption of soybean paste and other high nitrogen fermented products.

Abstract:[Background] Brucella canis is a pathogenic bacterium of canine brucellosis and mainly leads dogs to abortus and reproductive disorders. B. canis is a rare source of infection for human brucellosis, but safety risk of human posed by B. canis remains controversial. At present, the epidemiology characteristics and genetic diversity of B. canis research in China were lacking relatively. It is significant to investigate epidemiology characteristics and genetic diversity of canine brucellosis for strengthening surveillance and control of canine brucellosis. [Objective] Epidemiology characteristics and genetic diversity of B. canis strains were investigated in order to provide a reference for control and prevention of canine brucellosis. [Methods] Both conventional bio-typing methods and BCSS-PCR were applied for species identified of 63 B. canis strains. The genetic diversity of B. canis strains were investigated by the means of HGDI. MLVA method was used for cluster analysis of B. canis to reveal the epidemiological characteristics of canine with BioNumerics 5.0 software. goeBURST software was applied to construct minimum spanning tree of B. canis strains for elucidate characteristics of geographic origin of B. canis strains in China. [Results] Both conventional bio-typing methods and BCSS-PCR were used and 63 tested strains were all B. canis, and 100% identification coincidence rate was found between conventional bio-typing method and BCSS?PCR assay. The analytical sensitivity of BCSS-PCR at DNA sample was 10?3 (50 pg/μL DNA of detecting B. canis). The higher genetic diversity was observed in B. canis strains in China. The results of genetic diversity investigated have showed that five loci in Panel 2B presented higher variability, the orders of allele genotypes from high to low were bruce09 (11), bruce07 (8), bruce16 (7), bruce04 (6), bruce30 (5). MLVA cluster analysis showed that there were three small-scale outbreaks of canine brucellosis in Beijing and sporadic in other areas. B. canis strains in this study were divided into five geographic groups (I?V), MLVA-11 genotype 26 clone group was predominated population. The strains of this clone group had common geographic origin with the strains from countries such as the United States, Greece, Canada, France, Romania and Korea. The others four geographic groups were unique lineages of B. canis in China. [Conclusion] There were higher genetic diversity and extensive of geographic origin of B. canis strains observed in China, and the results exhibited characteristics of origin and evolution of co-existing of imported and native specific lineage.

Abstract:[Background] The disease, causing fulminant hemorrhagic of yellow catfish (Pelteobagrus fulvidraco), occurred in a pond in Dangtu County, Anhui Province, and the pathogen is controversial currently. [Objective] Investigate the pathogen causing outbreak hemorrhagic disease of yellow catfish (Pelteobagrus fulvidraco) and the biofilm formation characteristics of the isolated strain, to provide a reference for the prevention and treatment of A. jandaei infection. [Methods] The organs with obvious pathological changes in dying yellow catfish were inoculated on the EPC cell and media (TSB agar plate and blood agar plate) to isolate the pathogen, and the pathogenicity of the isolate was detected by artificial infection test. The isolated strain was identified by phenotypic identification and 16S rRNA gene sequence analysis. Then, the optimal conditions for biofilm formation, biofilm forming ability of the isolated strain and biofilm-forming genes carried by the strain were detected. [Results] A dominant isolated strain named HSY-2 was recovered and purified from organs with obvious pathological changes. In the artificial infection trail, the bacterial isolate HSY-2 exhibited virulence to yellow catfish with an LD50 value of 1.05×106 CFU/mL. The bacterial isolate was identified as Aeromonas jandaei according to morphology, biochemical characteristics and phylogenetic analysis derived from 16S rRNA gene. The optimum condition for forming biofilm was to inoculated on the TSB medium for 96 h at 30 °C, at which the isolated strain might form a moderate intensity biofilm. Three biofilm formation related genes, including glycerol-3-phosphate dehydrogenase D gene glpD, S-ribosylhomocysteine lyase gene luxS and LuxI family protein homolog gene ahyI were detected in the isolated strain, whereas the mannose-sensitive hemagglutinin pili biosynthesis protein Q gene was not detected. [Conclusion] This experiment provides a basis for further study on the regulation mechanism of A. jandaei biofilm formation and a reference for the prevention and treatment of A. jandaei infection from the perspective of anti-biofilm formation.

Abstract:[Backgroud] Porcine epidemic diarrhea, porcine rotavirus disease and pseudorabies are three important infectious diseases that seriously endanger the global pig industry. Mixed infections often lead to more serious losses on the farm. [Objective] A homologous recombination technique was used to construct a triple genetic engineering attenuated strain of pig pseudorabies co-expressing s gene and vp7 gene. [Methods] Through the sequence alignment and protein structure analysis of PEDV s and PoRV vp7 genes, 475?804 aa of s gene and 17?339 aa of vp7 gene were selected as candidate regions for the construction of strains. Three cloning vectors of pMD-S, pMD-VP7 and pMD-VP7.S and one transfer vector of pEGFP-VP7.S were constructed, respectively. Plaque purification was employed to obtain recombinant strain. [Results] A genetically engineered pseudorabies strain co-expressing S protein and VP7 protein was constructed. The strain was serially passaged for 20 generations, and both vp7 and s genes were detected without gE gene. Western blotting experiments confirmed foreign gene expression by recombinant virus. The TCID50 of the parent strain and the recombinant strain was determined by the Reed-Muench method to be 10?7.59/0.1 mL and 10?7.25/0.1 mL, respectively. [Conclusion] Recombinant pseudorabies strain PRV (CM) was obtained, which stably carried the two foreign genes, but with the deletion of virulence genes. The recombinant strain owned a similar proliferation characteristic with the parent strain. The study laid the foundation for further study on development of PRV, PEDV and PoRV triple genetic engineering vaccine.

Abstract:[Background] Avian pathogenic Escherichia coli (APEC) is one of the main pathogens of poultry. The quorum sensing (QS) system regulates its biological properties through signaling molecules. Currently, the effect of the signaling molecule AHLs on its biological properties in APEC is unclear. [Objective] To study the effect of AHL on the biological characteristics of APEC. [Methods] The plasmid containing the acyl-homoserine-lactone synthase (lasI) gene was transformed into the DE17 strain of APEC to construct the recombinant strain DE17-lasI. Then the biological characteristics, including AHLs production, growth characteristics, biofilm formation ability, motility and drug resistance was compared between DE17 and DE17-lasI, respectively. Besides, the transcriptional levels of virulence genes, biofilm formation related genes and flagellin synthesis genes of wild and recombination strain were compared by real-time PCR. [Results] The level of AHL produced by DE17-lasI was significantly enhanced (P<0.01), while the biofilm formation and motility were significantly decreased (P<0.01) compared with the wild strain DE17. However, the growth characteristics and drug resistance were no significant change (P>0.05) between DE17 and DE17-lasI. Furthermore, the results showed that the transcription level of virulence factor fimH of the DE17-lasI was up-regulated by 58.8 times, while ompA and iss were down-regulated by 95.4% and 77.3% times compared with DE17, respectively. The biofilm formation related gene agn43 and the flagellar synthesis gene flhA was down-regulated by 75% and 80.8%, respectively. In addition, the transcription level of the AHL receptor sdiA was up-regulated by 19.8-fold. [Conclusion] Transformation of lasI into APEC can promote the synthesis of AHL in APEC, which significantly affect some biological characteristics of APEC. This study provides a reference for further study on the regulation of AHLs in APEC.

Abstract:[Background] Aquaculture diseases are a significant constraint in the development of freshwater fisheries, and the microbial status of aquaculture environment is closely related to the health of fishes, which has drawn extensive attention. [Objective] To elucidate the change characteristics of pathogen abundances and bacterial community diversity in aquaculture environment. [Methods] real-time fluorescence quantitative PCR method and terminal restriction fragment length polymorphism (T-RFLP) technology were used to quantify typical pathogens in gibel carp breeding environment, and to analyze bacterial community diversity. [Results] Pathogen abundances in the process of breeding presented different change trend, the abundances of Aeromonas hydrophila, A. sobria and A. veronii were significantly positively related to Cyprinid herpesvirus Ⅱ. The abundance of pathogens in water was significantly affected by pH, temperature, dissolved oxygen and other environmental factors. T-RFLP analysis showed that the composition of bacterial community in sediment samples was more complex than that in water samples, and the dynamic change range of bacterial community in sediment was higher than that in water. The T-RFs in water and sediment ranged from 11 to 29 and 20 to 32, respectively, and Shannon-wiener index ranged from 1.44 to 2.87 and 2.44 to 3.25, respectively. [Conclusion] The abundances of pathogens in cultured waters were higher than that in sediments, and the relative abundance and diversity of bacterial community in sediments were higher than that in water. The changes of pathogen abundances and bacterial community structure are closely related to environmental factors, and it will provide theoretical data for guiding the early warning of pathogenic diseases in the breeding processes to clarify the ecological distribution and abundance changes of pathogenic microorganisms in the aquaculture pond.

Abstract:[Background] Protocatechuic acid is the main active ingredient of some plants and can be used as a precursor of many polymers as well as various drugs. At present, protocatechuic acid is mainly extracted from plants by chemical methods, with poor extraction efficiency and certain degree of damage to the environment. [Objective] The cloning and heterologous expression of ρ-hydroxybenzoic acid-3-hydroxylase gene ρ-HBA-3H for realizing the biotransformation of protocatechuic acid catalyzed by ρ-hydroxybenzoic acid-3-hydroxylase. [Methods] The ρ-hydroxybenzoic acid-3-hydroxylase gene ρ-HBA-3H was amplified by PCR using Rhodococcus sp. R04 genomic DNA as a template and then constructing recombinant genetic engineering strain BL21(DE3)/pET21a(+)-ρ-HBA-3H. The expression of ρ-hydroxybenzoic acid-3-hydroxylase was induced, and conducted biotransformated of PCA by this enzyme in the presence of the substrate ρ-hydroxybenzoic acid (ρ-HBA), besides, the conditions of biotransformation were optimized. [Results] The ρ-hydroxybenzoic acid-3-hydroxylase gene was highly expressed in E. coli. The yield of protocatechuic acid by biotransformation can reach 1.156 g/L. Optimization experiments showed that Mg2+ and Triton X-100 have no effect on the conversion efficiency. Increasing the dissolved oxygen content of the reaction system and adding appropriate amount of Tween- 80 can promote the conversion reaction. On the basis of continuous cell transformation, proper amount of glucose can effectively increase the transformation efficiency of engineering bacteria and reduce the consumption of protocatechuic acid. [Conclusion] This study realized the high-efficiency and green production of protocatechuic acid by bio-enzymatic catalysis, and provided theoretical research basis for the industrial production of other important fermentation products.

Abstract:[Background] Lactobacillus plantarum widely exists in many ecological environments, such as plants, dairy products, meat products and the intestines of mammals and insects. [Objective] To explore the potential link between genetic variability and environmental origin of L. plantarum isolates. [Methods] The genetic diversity and functional genome of 126 L. plantarum strains isolated from plants, dairy products, meat products, drosophila, mammals’ gut as well as mammals’ oral cavity and other locations, were deciphered by comparative genomic analysis to understand the genetic relationship and evolutionary history. [Results] The genomic size and number of coding genes of L. plantarum isolated from drosophila were significantly higher than those isolated from plants, mammals gut, meat products and dairy products (P<0.05), but there was no significant difference among plants, mammals gut, mammals oral cavity and other locations as well as meat products isolates. Both phylogenetic trees constructed based on single copy genes and core genes showed that drosophila isolates and dairy products isolates are clustered in a branch, respectively. while other isolates were evenly distributed into each branch. The results based on accessory genes analysis were consistent with the results based on the phylogenetic tree analysis. The analysis of functional genome showed that habitat-specific genes of drosophila isolates were involved in the metabolism of fructooligosaccharides and chitin, habitat-specific genes of dairy products isolates were involved in mazEF toxin-antitoxin system and CRISPR system. [Conclusion] L. plantarum strains isolated from drosophila and dairy products may have undergone adaptive evolution in order to adapt to the unique habitat. These results provided a new insight for the adaptive evolution and provided theoretical basis for analyzing the evolution process of L. plantarum.

Abstract:[Background] With the widespread use and even abuse of antibiotics, the problem of bacterial resistance has become increasingly prominent. The use of phage to treat drug-resistant pathogens has begun to attract attention. [Objective] Biological characteristics and bioinformatics analysis of a newly found phage vB_KpnP_IME279 of Klebsiella pneumoniae. [Methods] A multi-drug resistant Klebsiella pneumoniae was used as the host strain to isolate phage from hospital sewage. We used the double-layer plate method to conduct the titer, optimal multiplicity of infection (optimal MOI), one-step growth curve and lysis spectrum of the phage. Phage morphology was observed by transmission electron microscopy after purification. Its genome was sequenced using the Illumina MiSeq sequencing platform. Complete genome sequence was used for genome annotation, comparative genomics and evolutionary analyses. [Results] A novel phage vB_KpnP_IME279 was successfully isolated from infected host cells. The optimal MOI of IME279 is 0.1. One-step growth curve shows that IME279 has a burst size of 140 PFU/cell and a latent period of 20 min. The genome of IME279 is 42 518 bp and 59.3% (G+C)mol%. Electron microscopic observation showed that the phage was belongs to the family Podoviridae. The BLASTn alignment showed that the genome of the phage had limited similarity with the currently known phages. The evolutionary relationship between phage IME279 and other Podoviridae phage was analyzed by gene phylogenetic tree of phage major capsid protein, which suggests IME279 is a new member of the Podoviridae phage. [Conclusion] Isolation and identification of the new phage of Klebsiella pneumonia and the biological characteristics, genome-wide sequencing and bioinformatics analysis will help study the relationship between Klebsiella pneumoniae phage and host and the treatment of multi-drug resistant bacteria with phages.

Abstract:[Background] Adolescents acne is one of the most common chronic inflammatory dermatitis associated with abnormal proliferation of Propionibacterium acnes. [Objective] The aim of this study was to explore the difference of microbial composition between the acne skin and healthy control, and to provide theoretical basis for the prevention and treatment of acne from the perspective of microecology. [Methods] Bacterial 16S rRNA gene V1?V2 region sequencing and fungal TIS1 region sequencing technology were used to analyze the bacterial and fungal community structure of facial acne skin in 16-year-old teenagers in Beijing. Bacterial and fungal composition in adolescent acne with lesions and nearby areas without obvious lesions, as well as healthy controls, were investigated. [Results] Compared to the healthy samples, the skin bacterial diversity of adolescent with acne was significantly decreased (P<0.001), and the abundance of Propionibacterium (P. acnes) and Staphylococcus (Staphylococcus epidermidis PM221) decreased significantly. While there was no significant difference in the bacterial composition between the acne lesion area and the nearby no obvious lesion area. The fungal richness (Chao1 index) and the abundance of Malassezia restricta of the adolescent acne group was significantly higher than that of the healthy control group (P<0.05). [Conclusion] The change of skin microbial composition is associated with the occurrence of adolescents acne. This study provided theoretical basis for the prevention and treatment of acne from the perspective of microorganisms.

Abstract:Integrative and conjugative elements (ICEs) mediate bacteria bestow resistance to multiple antibiotics and some complex new traits through horizontal gene transfer (HGT). They encoded a wide variety of genetic information, including resistance to antibiotics and heavy metals and the capacity to degrade aromatic compounds which are affected by bacterial type IV secretion system (T4SS), the transfer of these genetic elements would speed up the dissemination of antibiotic resistance genes within and between microbial genera. It results in the growing problems of drug resistance, even multi-drug resistance and makes the mechanism of resistance become exceedingly complex. In this review, the structure and conjugation process of ICEs and the interactions with the components of T4SS are discussed.

Abstract:Iron is an essential nutrient for the survival of most bacteria and participates in many important life processes. Pathogens have evolved multiple mechanisms to acquire iron from the infected host. However, excess iron will produce cytotoxicity via the Fenton reaction; thus, iron concentration must be strictly regulated. In order to limit infection, the host has evolved a variety of strategies to restrict iron availability to invading microbes. Targeting microbial iron acquisition systems is therefore a promising antibacterial therapy strategy.

Abstract:Coastal wetland ecosystems, located at the boundary of land and sea, are characterized by high biodiversity, high primary productivity, active nutrient element cycling driven by microbes, and important sources of nitrous oxide (N2O) emissions. N2O is the third greenhouse gas only less than carbon dioxide (CO2) and methane (CH4), and it is estimated that microbes are responsible for more than 90% of global N2O emissions, which are closely associated with the diversity, composition and function of microbial communities involved in nitrogen cycling in coastal wetland ecosystems. This review focuses on microbially-driven N2O production and mechanisms that are coupled with carbon, sulfur and metal cycling, temporal and spatial patterns of N2O emissions regulated by environmental factors, and future research directions in coastal wetland ecosystems, aiming to reveal microbially-driven N2O production and regulatory mechanisms by environmental factors, and mitigate global warming.

Abstract:Poyang Lake, the largest freshwater lake in China, is a typical seasonal shallow lake. Its unique hydrological characteristics and various wetland landscape types make the microbial communities complex and diverse. This paper summarized the current research status of the microbes in Poyang Lake on: (1) effects of hydrological rhythm, nutrients and heavy metal contents on the composition of microbial community in Poyang Lake; (2) effects of water level elevation and wetland reclamation on soil microbial distribution in Poyang Lake wetland. Meanwhile, the future research direction of microorganism in lake wetland and the unique conditions of Poyang Lake are also discussed, providing an important reference for studying lake microorganisms in the future.

Abstract:Rapid depletion of reserved fossil fuel, this has made biomass-based bioenergy research becoming a research hotspot in the energy field in recent years. Making the most of renewable biomass can provide an excellent opportunity to develop economical biofuel production processes. Compared with ethanol and biodiesel, biobutanol has more advantages. Therefore, production of butanol from renewable lignocellulosic biomass has been widely studied in recent years. In order to obtain butanol from renewable biomass, some key scientific problems, such as the selection of raw material, low yield of product, toxicity of the inhibitor to the producing strain, need to be solved. In this study, the pretreatment of raw materials in the process of biobutanol fermentation with lignocellulose biomass as raw materials, the effect of inhibitors on butanol-producing strains, the detoxification of hydrolysate and the selection of strains resistant to inhibitors were reviewed. The opportunities and challenges in the production of butanol from lignocellulose were also discussed.

Abstract:With the rise and development of high-throughput sequencing technology, omics technology, bioinformatics technology, some achievements have been made in the study of the relationship between the two strains in vitamin C (Vc) microbial fermentation. Therefore, this paper reviews the current research on the companion mechanism between the two strains based on spores, amino acids, B-group vitamins, environmental stress and small molecular substances, and provides some new ideas for the further research.

Abstract:The problem of air pollution has of late years become increasingly sever, and industrial production is a momentous source. Biological treatments of gaseous pollutants including volatile organic compounds, odours, sulfides, nitrogen oxides and so forth in industrial production has gradually emerged in China and gained extensive attention with the advantages of high efficiency, low investment, green and environmental friendliness. This paper reviews fundamentals, technical classification and characteristics, applied range, state-of-the-art research directions and progress of biological treatments both here and abroad. Biological treatments tend to have high removal efficiency for the treatment of low concentration gaseous pollutants, while it is limited for treating hydrophobicity, high concentration, toxicity, and low bioavailability of these pollutants. To solve these problems, experts and scholars had done a great quantity of researches and experiments, and those efficient methods and means innovated were summarized. It is considered that the application of methods for reducing mass transfer resistance, combination with other treatment technologies, clarification of mechanisms and metabolic pathways of microbial degradation are significant research directions to improve the efficiency of biological treatments and expand its field of application. However, nearly all methods of intensification mentioned are still in the stage of experimental research, as an important goal, which still need to be continuously studied for industrialization.

Abstract:The global epidemic of tuberculosis caused by Mycobacterium tuberculosis (Mtb) is still grim. During host infection, Mtb secretes a series of effector molecules into human macrophage to regulate, modulate host immunization pathway and finally escape from the immune killing. Here, we summarized the critical roles of protein tyrosine phosphatase A (PtpA) during Mtb infection, such as inhibit the innate immunity, apoptosis, phagosome-lysosome fusion by interfering multiple pathways and modulate macrophage bioenergetics state. PtpA has been assumed as an important drug target for a long time. However, it is beset with the high similarity with human protein tyrosine phosphatase hLMW-PTP in inhibitor design and screening. At last, the research status and problems need to be revealed in Mtb PtpA transcriptional regulation and secretion pathway was analyzed and discussed, which will provide us a new way to block PtpA’s function.

Abstract:As a result of knowledge transferring before the class based on the flipped classroom, students can internalize their knowledge by accomplishing various interactive activities in class. Recently, we practiced the knowledge transferring of Immunology before class on the platform of “Wanke001.com”, which ensured that students can complete the task of self-study before class with high quality. This paper mainly studies the design, implementation and evaluation of Immunology classroom activities. This “teacher-led, student-centered” teaching model not only develops students’ self-learning ability, but also realizes the transformation of educational objectives from the primary stage of memorization and understanding to the advanced stage of application, analysis, evaluation and creation.

Abstract:To improve the quality of undergraduate education and implement the comprehensive and multi-dimensional education mode, we have taken the initiative of setting up a comprehensive design experiment of food microbiological detection module in our university since 2003. In this paper, the reform of practical teaching from the aspects of the course of opening the project, the key factors of project implementation, the primary focus of process education, the diversified quantitative assessment system, the feedback and reflection on teaching results were summarized. The practice results indicate that the module of food microbiology detection, as the feature and highlight of the course of microbiology experiment, had a good inheritance in the experimental preparation mode and scheme review mode. It plays an important role in cultivating students’ innovative spirit, scientific research quality, information quality, team spirit and data processing ability, etc. The special experiment mode is highly valued by teachers and students, which is unanimously recognized by peers. The establishment of diversified assessment system, such as process evaluation, promotes the teaching effect. The students have a higher evaluation on the experimental effect, who are satisfied with the learning results. In the next stage, this teaching mode can be further improved by strengthening the teaching team and cutting down learning burden of the students. The multi-dimensional and comprehensive talent training can be achieved in the modularization of food test experiment, which meets the current demand of “cultivating innovative, practical and comprehensive talents”, and therefore has a sound popularization value.