Abstract:[Background] The metagenome of sucrose-enriched soil is one of the important sources of sucrose phosphorylase. The enzyme has important applications in industries, as it can catalyze transglycosylation reaction to improve the properties of the receptors using cheap sucrose. [Objective] The properties and transglycosylation activity of sucrose phosphorylase isolated from the metagenome were studied, providing foundation and research materials for better modification. [Methods] A gene encoding sucrose phosphorylase from metagenomic library was cloned into the expression vector pSE380, and the recombinant strain was constructed. Then, the target protein was purified by nickel affinity chromatography. The properties were determined using sucrose as substrate, and transglycosylation activities of the carbohydrate receptors were also investigated. After protein modeling and substrate channel were analyzed, the site-saturation mutagenesis at the amino acid 155 was performed by reverse PCR. The enzymatic properties and transglycosylation activity of the mutants also were studied in detail. [Results] The molecular weight of purified protein is about 56 kD. And the recombinant protein exists in the form of a trimer in the active state. The optimum temperature and pH value were 55 °C and 6.5, respectively; Km and Vmax value were 23.1±2.4 mmol/L and 407.9±8.5 μmol/(mg·min) using sucrose as substrate. Among the site-directed saturation mutants of the amino acid 155, some mutants showed improved enzymatic properties or transglycosylation activities. [Conclusion] The study of sucrose phosphorylase from the metagenomics enriched the enzymatic data. The mutants with better properties were obtained, which provides theoretical foundation for researching on key amino acids related to transglycosylation and the applications of sucrose phosphorylase.

Abstract:[Background] At present, antibiotics are mainly used to control pathogenic bacteria such as Vibrio harveyi in seawater breeding industry. However, the long-term application or abuse of antibiotics may be adverse to the environment protection and human health. Therefore, biological control methods that are both environmentally friendly and effective have broad application prospects. [Objective] Strains with antibacterial activity were screened from the symbiotic microorganisms of seawater products. The active strains were identified and the culture conditions for the synthesis of antibacterial active substances were determined. [Methods] Fungi and bacteria were isolated from mariculture animals by the dilution and spreading plate method using the culture medium of Sabouraud and 2216E medium. The antibacterial activity of microbial fermentation broths against V. harveyi was determined by Oxford cup method. The active strain was identified by the morphological characteristics and internal transcribed spacer (ITS) analysis. Culture conditions were determined by screening the type and salinity of fermentation medium. [Results] A total of 52 microbial strains were isolated from 9 kinds of samples, among them, 30 strains belong to fungi and 22 strains belong to bacteria. Two fungal strains with antibacterial activity against V. harveyi were obtained. One active strain, HLZ-3, was identified as Aspergillus tubingensi. The culture conditions of HLZ-3 for the synthesis of antibacterial active substances was 4% NaCl in rice culture medium, and stationary incubated at 28 °C for 2 weeks. [Conclusion] The results provide a basis for further isolation and purification of antibacterial secondary metabolites produced by the strain HLZ-3.

Abstract:[Background] Rehai hot springs in Yunnan Province contain a wealth of extreme microbial resources. [Objective] This study was proposed to study the microbial diversity and community structures in Rehai hot springs, especially the diversity of iron- and sulfur-oxidizing microorganisms in the acid hot springs. [Methods] The microbial 16S rRNA V4?V5 genes region was sequenced by Illumina HiSeq technology and analyzed by bioinformatics software. [Results] A total of 578 061 high-quality sequences were obtained from 15 samples of 3 hot springs, resulting in 141 OTU belonging to 19 phyla, 66 genera. Crenarchaeota and Firmicutes were main phyla in Gumingquan (GMQ), Hamazui (HMZ) and Huangguaqing (HGQ). On the genera level, there were 37 and 32 genera in alkaline hot spring GMQ and neutral hot spring HMZ, respectively, in which the major genera were Bacillus and Pyrobaculum. Among the 20 genera present in acidic hot spring HGQ, the genera Acidibacillus and Acidithiobacillus were dominant. Furthermore, some species of Acidicaldus, Sulfobacillus, Sulfolobus and Metallosphaera genera were isolated from HGQ, which have potential to oxidize iron and sulfur. [Conclusion] The microbial resources were diverse in Rehai hot springs and the microbial compositions of the three hot springs were obviously different. Iron- and sulfur-oxidizing microorganisms, uncultured and unclassified microbes, especially archaea, were rich in acidic HGQ.

Abstract:[Background] The bioconversion of cellulose has become a research hotspot in the energy, food and chemical industries. However, there are few reports on the cold-adapted fungi with the ability to degrade cellulose. [Objective] To isolate and screen the cold-adapted fungi with high efficiency of cellulose degradation from the rhizosphere soil in the high altitude area of Tibet, and optimize their enzyme production conditions, so as to lay a foundation for their industrial application. [Methods] Cellulolytic strains were screened by the dilution plate coating method, the qualitative analysis of Congo red and the quantitative analysis of enzyme activity. Strains were identified by morphology and ITS rDNA sequence analysis. The fermentation condition was optimized by single factor and response surface methodology. [Results] A cold-adapted fungus, namely NLS-2, was obtained and identified as Penicillium sp. At 15 °C, the optimized culture conditions of strain NLS-2 for producing cellulase were straw powder 2.5%, yeast powder 0.5%, KH2PO4 0.5%, fermentation time 7 d, pH 6.5, shaker speed 170 r/min. [Conclusion] Penicillium sp. NLS-2 can grow at low temperature and efficiently produce cellulose, which has a good application prospect.

Abstract:[Background] The problem of heavy metal cadmium (Cd) pollution is becoming increasingly serious. Microalgae is a good biological adsorbent of heavy metals. However, current researches have mainly focused on the Cd removal rate and adsorption performance of microalgae, while their anti-Cd mechanisms were seldom studied. [Objective] In our study, the effects of Cd stress on the physiological character, resistance mechanism and oil productivity of oleaginous microalgae Auxenochlorella protothecoides UTEX 2341 were studied. [Methods] The growth and oil productivity of microalgae under 0?5 mmol/L Cd stress were measured. Changes in pigment, soluble protein and oil content of algae under 2 mmol/L Cd stress were further analyzed, as well as changes in their submicrostructure, antioxidant enzymes, antioxidants and fatty acid components. [Results] Auxenochlorella protothecoides UTEX 2341 could withstand 2 mmol/L of Cd stress. Its biomass and chlorophyll content slightly decreased, while its lipid yield significantly increased to 1.60 g/L at 168 h, 1.77 times of the control. Besides, high Cd treatment induced the accumulation of reactive oxygen species (ROS). In response to the oxidative damage caused by high Cd stress, the microalgae cells initiated their resistance mechanism, as the content of antioxidants of carotenoid and reduced glutathione (GSH) remarkably increased to 1.42 and 4.5 times of the control, alleviating the toxicity of Cd, though the activity of antioxidant enzymes was suppressed by Cd stress. According to the results of fatty acid composition analysis, the content of C18:1 increased and the percentage of C16?C18 reached 96%?98%, meeting the production standard of biodiesel. [Conclusion] This research laid a foundation for future researches on the anti-Cd mechanism of microalgae and the algae lipid synthesis regulation mechanism under Cd stress.

Abstract:[Background] China is a large aquaculture country. Ammonia nitrogen and nitrite are the main nitrogen source pollutants in water. Excessive ammonia nitrogen in water can damage the nervous system, liver and kidney system, body surface and internal organs filled with blood of aquatic animals. Excessive nitrite reduces the ability of carrying oxygen by red blood cells, and immune power of fish and shrimps, results in various diseases, and even suffocation. Some photosynthetic bacteria with the function of removal of ammonia nitrogen and nitrite in water are environmental friendly. [Objective] The photosynthetic bacterium strain (No. SP3) isolated from the mixed bacteria samples from aquaculture ponds in Guangdong province will be identified for its scientific species and removal ability of ammonia nitrogen and nitrite in water so as to provide the target bacterial strain for the biological removal of the water body. [Methods] The photosynthetic bacterium strain (SP3) was isolated from mixed bacteria samples from aquaculture ponds in Guangdong province using double-layer plate method. The strain SP3 was identified by 16S rRNA gene sequence analysis, gram staining, carbon source utilization and inorganic electron donor utilization experiments. The optimal culture conditions of strain SP3 were determined by detection of OD600 in different pH and various NaCl concentrations. The utilization of strain SP3 for different nitrogen sources (amonium chloride, sodium nitrite) could be confirmed by measuring the change trends of OD600 in different concentrations of the two nitrogen sources during the period of 7 days. The ability of strain SP3 to get rid of ammonia nitrogen and nitrite in water were determined by Nessler?s reagent colorimetric method and spectrophotometry. The nitrite reductase gene (nirS) was cloned by means of Genome Walking method, and the expression dynamics of nirS in the removal process of ammonia nitrogen and nitrite nitrogen were studied by real-time RT-PCR. [Results] The isolated strain SP3 is Gram-negative, short rod-shaped, and can utilize acetate, pyruvate, pyruvate, propionate, butyrate, lactate, fumarate, succinic acid, malate, fructose and glucose as carbon sources, but can not use ethanol and propanol as carbon sources. It can employ Na2S2O3·5H2O, Na2S·9H2O and Na2SO3 as inorganic electron donors. The strain SP3 is closely similar to Ectothiorhodospira shaposhnikovii (99% similarity) based on the 16S rRNA gene sequence analysis. The suitable pH is 6.0–8.5, and the suitable salinity is 0–3% for SP3. The growth status of strain SP3 using ammonia nitrogen chloride as a single nitrogen source was significantly better than that using sodium nitrite as a single nitrogen source. Under the condition of an initial bacterial concentration of 8.6×109 cfu/mL, the ammonia nitrogen at an initial concentration of 84.15±0.58 mg/L was removed in water for 7 days was 79.45±0.29 mg/L, and the removal percentage reached 94.42%, meanwhile, the sodium nitrite at an initial concentration of 2 mg/L degraded in water for 5 days was lower than the detection limit (0.003 mg/L). The relative mRNA expression level of nirS in the strain SP3 was upregulated during the period of removal of ammonia nitrogen and nitrite. [Conclusion] The strain SP3 was identified as E. shaposhnikovii, and had the strong ability of removing ammonia nitrogen and nitrite. Therefore, it is a promising photosynthetic bacterium strain in application of aquaculture water and wastewater treatment.

Abstract:[Background] Although clodinafop-propargyl (CP) can efficiently control malignant weeds in wheat field, the producing and using of CP cause damage to the environment, and pose a threat to the health of animal and human. [Objective] We isolated an efficient CP-degrading bacterium and studied its degradation characteristics to provide microbial resource for bioremediation of CP pollution. [Methods] The active sludge sample was collected from a pesticide factory. The CP-degrading bacterium was isolated by enrichment cultivation and Luria-Bertani (LB) medium containing CP, and identified by morphological, physiological characteristics and analysis of 16S rRNA gene sequence. Single factor experiments were used to evaluate the degradation characteristics including temperature, pH, inoculum, substrate concentration. The degradation product of CP was identified by UPLC-MS. [Results] An efficient CP-degrading strain WP68 was isolated and identified as Sphingopyxis sp.. Strain WP68 was able to degrade 98.26% of 200 mg/L of CP within 10 h under the condition of 37 °C and pH value of 8.0. The optimal culture conditions for strain WP68 degrading CP were temperature 37 °C, pH 8.0, inoculum 5%, and substrate concentration 200 mg/L. The degradation product of CP was identified as clodinafop acid (CA) by UPLC-MS. Strain WP68 was also capable of degrading cyhalofop-butyl and quizalofop-p-ethyl. [Conclusion] Based on the high efficient degradation ability and high CP-tolerance, Sphingopyxis sp. WP68 has the potential prospect in bioremediation of CP-contaminated soil.

Abstract:[Background] Endophytic diazotrophic bacteria can colonize within plants to provide nutrients for plants and promote plant growth through metabolism. [Objective] We studied the diversity of endophytic diazotrophic bacteria isolated from Bryophyllum pinnatum. [Methods] Endophytes were isolated and purified from surface sterilized plant tissues. Nitrogen-fixation ability of the isolates was tested by acetylene reduction assay. SDS-PAGE patterns of the whole-cell protein electrophoresis and IS-PCR DNA finger-printings were used to group these isolates. The representative strains were identified by physiological and biochemical characteristics, and the 16S rRNA gene sequence analysis. The growth promotion of nitrogen fixation, secretion of auxin and ACC deaminase, siderophore production, solubilizing phosphorus and potassium were determined. [Results] A total of 26 endophytic diazotrophic bacteria were isolated from Bryophyllum pinnatum and classified into 5 groups, belonging to 5 species of 4 genera. All representative strains had various plant growth promoting activity. [Conclusion] Endophytes isolated from Bryophyllum pinnatum have abundant genetic diversity and growth promoting characteristics.

Abstract:[Background] Tomato grey mould caused by Botrytis cinerea is an important fungal disease. Biological control can be an effective measure in prevention and control of this disease, due to its environmentally-friendly and not easy to develop pathogen resistance characters. [Objective] To screen antagonistic Streptomyce sp. with broad-spectrum antifungal activity, confirm the biocontrol effects and define the classification status of the antagonistic strain. [Methods] Antagonistic actinomycetes were obtained by agar block method, the inhibition spectrum of strain T22 was detected using confrontation culture and mycelia growth assays. Biocontrol effects of strain T22 was detected using enzymes production, detached leaves and seed germination assays. Strain T22 was identified to species level based on morphological and cultural features, physiological-biochemical characters, as well as molecular methods. [Results] A total of 14 antagonistic isolates were screened from 56 soil actinomycetes, among which strain T22 showed obvious inhibition effect on Botrytis cinerea and other fungi. The inhibition rate of strain T22 on Botrytis cinerea, Monilinia fructicola and Fusarium oxysporum f. sp. cucumerinum were 84.6%, 81.5% and 79.1%, respectively. In addition, the culture filtrate of strain T22 displayed obvious disease reduction effect (55.1%) on detached leaves of tomato plants. The hypocotyl length, radical length and seed vigor index were increased by 15.1%, 29.7% and 43.9%, respectively, with 100-fold dilution treatment. Strain T22 was identified as Streptomyces alboniger based on morphological and cultural features, physiological-biochemical characters, as well as multiple sequences analyses. [Conclusion] Streptomyces alboniger T22 had obvious antifungal activity, extracellular hydrolase production ability, disease reduction and seed germination promotion effects on tomato, with application potential as biocontrol agent against tomato grey mould.

Abstract:[Background] Bacillus licheniformis TG116 isolated from Typhonium giganteum Engl is a biocontrol strain with broad-spectrum against plant pathogens. [Objective] In order to optimize the enzyme production conditions of TG116 and explore its enzymatic properties. [Methods] The optimization of enzyme production conditions and the enzymatic properties of proteases were investigated by Folin-Phenol chromogenic method and response surface methodology. [Results] The optimal enzyme conditions to produce strain TG116 were temperature 40.83 °C, pH 8.01, and fermentation time 53.74 h and increasing aeration could significantly enhance enzyme activity. After 48 hours of incubation under optimized conditions, the protease activity of the supernatant reached 254.07 U/mL from 57.46 U/mL. The protein was alkaline protease, with optimum pH 8.5, the optimum temperature 50 °C and it has good temperature and pH stability. EDTA had strong inhibitory effect on enzyme activity and metal ions Mg2+, Ca2+, Na+, Co2+, K+ also have showed a certain inhibitory effect. [Conclusion] Strain TG116 has great pH and temperature stability. The proteases are not easily inactivated in practical applications, which can decompose the cell wall protein components of fungi and destroy the cell wall structure, thus inhibiting and even killing pathogenic bacteria to achieve antibacterial effects.

Abstract:[Background] Xinjiang is the largest salinized soil distribution area in China. Soil salinization inhibits plant growth seriously. Salt-tolerant promoting bacteria can improve soil fertility, crop stress resistance and soil utilization effectively, thus it promotes plant growth. [Objective] We isolated and screened salt-tolerant strains from rhizosphere soil of Suaeda dendroides. The strains with excellent growth-promoting effect were identified, and the microbial resources were excavated. [Methods] The traditional method of separation was used to screen the salt-tolerant bacteria in the rhizosphere of S. dendroides. The strains of salt-tolerant promoting bacteria were obtained through the three-stage screening system. DNA of the strains was extracted by CTAB method, and the 16S rRNA gene sequencing was performed for phylogenetic analysis to determine the classification status of the salt-tolerant bacteria. [Results] A total of 58 salt-tolerant bacteria were isolated from the rhizosphere of S. dendroides. There were 8 strains with nitrogen fixation activity, 12 strains with phosphate solubilizing activity, and 15 strains with potassium solubilizing activity, 3 strains with IAA activity, and 2 strains with strong ammonia producing ability. Strains named GTZW50-5 and MH-F promoted the growth of Arabidopsis thaliana significantly. The results of wheat pot experiment showed strain GTZW50-5 had significant growth promotion effect on root length and plant height of wheat, and to some extent, the chlorophyll content in plants was improved. Strain MH-F had a significant promoting effect on the roots of wheat, and the chlorophyll content and proline content of wheat were increased at different salt concentrations. According to phylogenetic analysis, the similarity between GTZW50-5 and Bacillus vallismortis (AY603658) was 99.43%, GTZW50-5 was identified as Bacillus vallismortis. The similarity between MH-F and Enterobacter ludwigii (JTLO01000001) sequence was 98.34%, which was identified as Enterobacter. [Conclusion] Strains GTZW50-5 and MH-F had a good effect of promoting growth, providing a theoretical basis for the development and utilization of salt-tolerant microbial resources.

Abstract:[Background] As an important group of soil microorganisms, bacteria can effectively promote the material cycle and energy flow of soil. Bacterial diversity and community structure can reflect the quality of soil. High-throughput sequencing has been widely used in studying soil microorganisms, and can get the classification information of soil bacteria more conveniently and accurately than traditional sequencing technology. [Objective] To analyze the bacterial diversity in alfalfa soil of drip irrigation as well as to study the relationship between the bacterial diversity and environmental factors. [Methods] High-throughput sequencing on PCR-ampllified 16S rRNA gene V3?V4 fragments was used to determine the bacterial diversity in rhizosphere and non-rhizosphere soils of alfalfa under two growth patterns, and were analyzed and compared. [Results] The richness of bacterial communities calculated by the Chao1 index and the bacterial community diversity calculated by the Shannon index showed the same trend: drip irrigation alfalfa soil>natural rainfall alfalfa soil; 879 675 high-quality sequence were derived, 4 431 operational taxonomic units (OTUs) from 4 groups of soil samples, which were classified into 46 categories, 53 classes, 116 orders, 220 families and 469 genera, except some unidentified bacteria. Proteobacteria (25.27%?34.42%) was the dominant phylum, of which Alphaproteobacteria (11.41%?18.97%) was the dominant subgroup and Sphingomonas (1%?4.54%) was the dominant genus. Compared with natural rainfall, the community structure of 6 phylum and 16 genus of rhizosphere soil bacteria under drip irrigation changed significantly. In addition, RDA analysis showed that different environmental factors had different effects on the microbial community. The abundance of 9 bacteria genera in the rhizosphere soil of drip irrigation was positively correlated with total phosphorus, available phosphorus, available nitrogen, total potassium, organic matter, soild-neutral phosphatase and soild-urease contents. [Conclusion] As a new water-saving technology, drip irrigation increases the diversity and abundance of bacteria in rhizosphere soil of plants on the basis of promoting plant growth, increasing yield and saving cost. The results provide scientific data for the reform of new irrigation systems and the development and utilization of soil microbial resources.

Abstract:[Background] Discovery and utilization of microorganisms with biocontrol activity is important to keep crop health and yield in agricultural production. [Objective] Our study aimed to clarify the taxonomic status of Bacillus sp. SK007 isolated from soil, and to validate its antagonistic effect on multiple plant pathogens and, therefore uncover its potential biocontrol functions. [Methods] We first used 16S rRNA gene and whole-genome sequencing analysis to determine the taxonomic status of strain SK007. Plate isolate-antagonistic experiments were applied to explore the antagonistic effect of strain SK007 on Botrytis cinerea, Alternaria brassicicola, Alternaria alternata, Fusarium graminearum and Fusarium solani. AntiSMASH was used to predict antibiotic genes of strain SK007. [Results] Based on the analysis of 16S rRNA gene and whole-genome sequencing, average nucleotide identity and digital DNA-DNA hybridization, the results demonstrated that strain SK007 belonged to Bacillus velezensis, and possessed the genes in producing the lipopeptide antibiotics and polyketone antibiotics. In addition, SK007, with a large number and high abundance of antibiotic gene clusters in genome, exhibited strong resistance to a variety of plant pathogenic fungi. [Conclusion] Bacillus velezensis SK007 has excellent traits in plant disease resistance, and thus has potential roles in facilitating plant resistance to disease and promoting crop yield.

Abstract:[Background] With the expansion of potato planting area, the occurrence of potato scab disease becomes more and more serious. Chemical control of potato scab caused a serial of problems like environmental pollution. Therefore, biological control with safety and efficiency becomes the focus of the current research. [Objective] To isolate and identify an antagonistic strain against Streptomyces scabies, and analyze its functional genes. To isolate the antimicrobial?substance and characterize its antimicrobial features. [Methods] The antagonistic strain was screened and identified from the soil with potato scab disease, and the functional genes were analyzed by PCR. The antimicrobial?substance was separated by ammonium sulfate precipitation method, and its antimicrobial spectrum and stability were tested. The biocontrol effect of the antagonistic strain was further verified by pot assay. [Results] An antagonistic strain BU396 was obtained by antimicrobial tests, which was identified as Bacillus velezensis according to its morphological characteristics, physiological and biochemical properties and molecular identification results. Functional genes analysis showed that BU396 contained four antimicrobial peptide synthesis related genes. The antimicrobial substance was precipitated from the supernatant of culture medium by ammonium sulfate precipitation with 75% saturation. The antimicrobial spectrum test showed that the antimicrobial substance could inhibit several plant and animal pathogens. The stability tests showed that the antimicrobial substance had good stability with thermal, protease, metal ions and wide pH value. Pot assay result showed that the incidence of potato scab was significantly reduced by BU396 treatment. [Conclusion] In this study, Bacillus velezensis BU396 with significant antagonism against Streptomyces scabies was isolated and identified, which was further detected containing several antimicrobial peptide related genes. The antimicrobial substance of BU396 had broad-spectrum antimicrobial activity and good stability. Moreover, it was also proved that BU396 had remarkable biocontrol effect against potato scab. This study lays the foundation for biological control of potato scab and further study of antimicrobial substances.

Abstract:[Background] At present, marker gene gfp has become an important tool to study the interaction between target microorganism and host. Using gfp to mark the biocontrol strain, we can track the survival and colonization of the biocontrol strain effectively. [Objective] The colonization of Ahn75 on rice was studied by making fluorescence labeling in the biocontrol actinomycete Ahn75, to lay a foundation for the resistant mechanism study of Ahn75 against rice blast. [Methods] Plasmid pIJ8655 with green fluorescent marker gene (gfp) was introduced into E. coli ET12567 by electroporation, then the gfp gene was incorporated into the genome of Ahn75 through conjugal transfer. The inhibitory activity of Ahn75-GFP against rice blast pathogens was tested by the dural culture method. Ahn75-GFP colonized in rice by spraying spores was observed by fluorescence microscopy, and re-isolated from the rice and counted, to explore the distribution of Ahn75-GFP in rice tissues. [Results] The PCR amplification of gfp and the fluorescence observation of Ahn75-GFP showed that, the green fluorescent marker gene was successfully integrated into Ahn75. The inhibitory activity of Ahn75-GFP against rice blast pathogens by the dural culture method was not significantly different from the original strain. Ahn75-GFP colonized in the root, stem and leaf, could be observed by a fluorescent microscope, and the re-isolation results of endophytic strains in rice indicated that the strains had the strongest colonization ability in stems. [Conclusion] A green fluorescence-labeled biocontrol strain Ahn75-GFP was successfully obtained, and showed good colonization ability in rice, which was of great significance for studying the control of rice blast by Ahn75 strain.

Abstract:[Background] In recent years, the production technology of sesame-flavor liquor is becoming more and more mature. But the corresponding scientific research has not developed synchronously. High-throughput sequencing technology is increasingly used in the study of species diversity, but it focuses on the relative abundance of species rather than the microbial number of species. [Objective] The changes of microbial community structure and its correlation with stress factors during the fermentation of sesame-flavor liquor were analyzed, and the correlation between main yeasts and bacteria was also studied, to provide theoretical support for revealing the fermentation mechanism and controlling fermentation quality of sesame-flavor liquor. [Methods] The microbial community structure during the fermentation of sesame-flavor liquor was determined by traditional quantification of microorganisms and sequencing the bacterial 16S rDNA gene and the fungal ITS rDNA gene using Thermofisher?s Ion S5TMXL sequencing platform. The contents of lactic acid, acetic acid and ethanol during the fermentation process were monitored. The microbial community and its relationship with stress factors during fermentation were analyzed by complexity analysis of samples, comparative analysis of samples and correlation analysis of environmental factors. The correlation between yeasts and bacteria was analyzed by Pearson correlation. [Results] Cellulose bacteria, Westermania and Bacillus were dominant in the early fermentation stage, and Lactobacillus was dominant in the middle and late fermentation stages, followed by cellulose bacteria, Westermania and Bacillus. Issatchenkia was the dominant yeast in the whole fermentation process, followed by Wickerhamomyces, Saccharomyces cerevisiae and Candida. Most microorganisms were negatively correlated with stress factors, and only Lactobacillus showed a significant positive correlation with acetic acid. Yeasts were positively correlated with some bacteria. [Conclusion] The interaction of stress factors and microbes in liquor fermentation process promoted the succession process of the community. The organic acids produced by Lactobacillus and Bacillus during the late stage of fermentation inhibited most of the acid-nonresistant microorganisms, and organic acids were the main stress factors affecting community structure. The combination of microbial quantity and relative abundance revealed more information about the succession of community structure and its correlation with environmental factors during fermentation.

Abstract:[Background] Aeromonas schubertii is a gram-negative short rod-shaped bacterium. It is a human-animal-fish co-infected conditional pathogen and can cause various human diseases, which has been reported frequently in recent years. [Objective] In order to provide references for the prevention and control of A. schubertii caused diseases in aquaculture , the physiological and biochemical characteristics, pathogenicity as well as the sensitivity to common antibiotics of the 4 strains of A. schubertii (WL-4, ZL-1, ZL-13, D075) isolated from hybrid snakehead have been analyzed and compared. [Methods] The physiological and biochemical characteristics of the strains were detected by automatic microorganism identification instrument, the gyrB and 16S rRNA gene sequences of the strains were compared by PCR and sequencing. The expression of virulence factor was tested by modified plate method and the carrying status of virulence gene and antibiotic resistance gene were detected by PCR. The differences of pathogenicity for 4 strains were analyzed by artificial injection infection. The minimum inhibitory concentration of 15 antimicrobial agents against the strains were measured by double microdilution method. [Results] As ATCC43700, a human origin strain, was used as a reference strain, the physiological and biochemical characteristics of these 5 A. schubertii were basically the same and the similarity of the gyrB and 16S rRNA genes of them were 99.57% and 100% respectively. five strains are clustered into one group though cluster analysis. All of them showed hemolytic activity, protease activity and lipase activity, but there are differences on virulence genes among 5 strain. ATCC43700 carried hlyA, ela, lip, ahal and act, while WL-4, ZL-1, ZL-13 and D075 carried hlyA, ela, lip, ahal and aer. four strains isolated from snakehead hybrid showed different pathogenicity. The mortality rate of hybrid snakehead by artificial injection of WL-4, ZL-1, ZL-13 and D075 at 1.2×105 CFU/mL solution was 30%, 40%, 70% and 80% respectively. The antibiotic sensitivity and antibiotic resistance gene are also different among them. [Conclusion] There are differences on pathogenicity and antibiotic sensitivity among the four strains of A. schubertii from hybrid snakehead. The results of this study are helpful to further research on the pathogenesis of A. schubertii and the prevention and control methods against it in aquaculture.

Abstract:[Background] Microbial natural products have become an important source of small molecule drugs and prodrugs in recent years. Genomic analysis shows that Streptomyces antibioticus NRRL 8167 contains a variety of biosynthetic gene clusters of natural products, with the potential to generate multiple new compounds. [Objective] Study on the secondary metabolites in S. antibioticus NRRL 8167 to discover compounds with novel structures or unique biological activities, and further explore the corresponding biosynthetic gene clusters. [Methods] Using HPLC-MS and UV spectrum, we excluded the known compounds produced by S. antibioticus NRRL 8167 and identified an unknown compound with special UV-vis absorption as isolation targets. The secondary metabolites were isolated and purified using normal or reverse phase column chromatography and high-performance liquid chromatography (HPLC). The structures of the compounds were elucidated by using mass spectroscopy (MS) and nuclear magnetic resonance (NMR). The genomic DNA of S. antibioticus NRRL 8167 was extracted for genome sequencing by the PacBio sequencing platform. Bioinformatics technique was used to annotate the genome, to define the gene clusters that is responsible for the compound biosynthesis, and to propose its biosynthetic pathway. [Results] The compound was purified and identified as naphthgeranine A, belonging to polyketides. The whole genome sequence contains 28 secondary metabolites biosynthetic gene clusters. The gene cluster 20 is proposed to be responsible for the biosynthesis of naphthgeranine A. We also speculated the biosynthetic pathway of naphthgeranine A. [Conclusion] Naphthgeranine A was purified from S. antibioticus NRRL 8167 and structurally elucidated based on the UV-vis spectra, MS data and NMR. Complete genome sequence of this strain provided the opportunity for defining the gene clusters of naphthgeranine A. The proposed biosynthetic gene cluster and biosynthetic pathways laid the foundation for further research on the biosynthetic mechanisms of naphthgeranine A.

Abstract:[Background] Auxotrophy is a widely applied biomarker in production and scientific research, but there is no report on Ganoderma lucidum. [Objective] This study was conducted to provide parent strains and technique supports for genetics research, cross breeding and spawn identification of Ganoderma lucidum. [Methods] The uracil auxotrophic mutant strains were obtained by UV mutagenesis of protoplast. The randomly selected uracil auxotrophic monokaryon strains were used as parents, and crossed with each other by Monkaryon-monkaryon mating to establish uracil auxotrophic dikaryons. [Results] eight uracil auxotrophic mutant strains were obtained by UV mutagenesis of protoplast. seven uracil auxotrophic dikaryon strains were obtained by Monkaryon-monkaryon crossing. [Conclusion] The testing results showed these auxotrophic strains restored their growth on PDA medium with uracil. The uracil auxotrophic dikaryon strains would be used as a tool for genetic transformation study and breeding.

Abstract:[Background] The emergence and dissemination of new mobile antibiotic resistance genes such as ndm and mcr, poses a great threat to the treatment of infections associated with multi-drug resistant Gram-negative bacterial pathogens. [Objective] To screen new antibiotics, bacterial candidates were isolated and characterized against Gram-negative bacteria. [Methods] First, bacterial strains were isolated from soils using tryptic soy agar (TSA), and identified by 16S rRNA gene sequencing. Then, bioinformatics analysis was performed based on the whole genome. Meanwhile, the secondary metabolites of the candidate strains were predicted by antiSMASH and antibacterial activity was confirmed by the double layer agar plate assay. Finally, the crude was extracted in methanol and identified using HPLC-MS/MS. [Results] Paenibacillus pabuli CAU136 was isolated from the soil in Beijing, China. The whole genome sequence and antiSMASH analysis showed the great potential of P. pabuli CAU136 to produce secondary metabolites against bacteria. P. pabuli CAU136 showed antibacterial activity against different Gram-negative bacterial strains using the double layer agar plate method. Colistin in the crude extracts of P. pabuli CAU136 was demonstrated based on HPLC-MS/MS analysis. [Conclusion] P. pabuli CAU136 may produce colistin against diverse Gram-negative bacterial strains.

Abstract:[Background] Salmonella typhimurium is one of the major intestinal pathogens. The use of probiotics to treat intestinal pathogen infection has becoming a new, green micro-ecological method. [Objective] Investigate the antibacterial activity and mechanism of the cell-free supernatant (CFS) of Bifidobacterium breve on S. typhimurium in vitro. [Methods] The microdilution method was used to detect the minimum inhibitory concentration (MIC) and sub-inhibitory concentration (SIC) of YH68 CFS against S. typhimurium. The antibacterial mechanism of YH68 CFS against S. typhimurium was investigated from the changes of cell morphology, cell membrane permeability, membrane integrity and virulence gene expression of S. typhimurium. The adhesion and invasion of S. typhimurium to HT29 cells were also studied. [Results] YH68 CFS (3×109 CFU/mL) has a good inhibitory effect on S. typhimurium, and the diameter of the inhibition zone is 22.27±0.44 mm. When YH68 CFS was at MIC concentration (250 μL/mL), its mechanism of action against S. typhimurium was to increase the membrane permeability, destroy its integrity, form the cell membrane pores, and ultimately achieve antibacterial purposes. However, YH68 CFS did not affect the growth of S. typhimurium at the concentration of SIC (62.5 μL/mL), but it could still inhibit the adhesion and invasion of intestinal epithelial cells by down-regulating the expression of its virulence genes. [Conclusion] Bifidobacterium breve YH68 has a good antibacterial effect against S. typhimurium and could be used as a potential probiotic for clinical treatment of Salmonella infection.

Abstract:[Background] Pseudomonas aeruginosa (P. aeruginosa) specifically interacts with (oxidized) low density lipoprotein (LDL/oxLDL), and RahU expressed by P. aeruginosa binds to LDL/oxLDL. [Objective] Our study investigated if RahU on P. aeruginosa was the major binding ligand for LDL/oxLDL. [Methods] The interaction of recombinant RahU (rRahU) with LDL/oxLDL was detected by ELISA. Mouse anti-rRahU antibody was generated to determine if RahU was expressed on the membrane of the wild P. aeruginosa strain by Western-Blotting and ELISA. The affinity of LDL/oxLDL bind to wild P. aeruginosa strain and ΔRahU strain (constructed as a negative control) was compared by ELISA. Influence of different protease digestion on the binding affinity of ΔRahU strain to LDL/oxLDL was also compared by ELISA. [Results] Recombinant RahU specifically interacted with LDL/oxLDL. Western-Blotting and ELISA results revealed that anti-rRahU antibody bound to the membrane protein extracted from wild P. aeruginosa strain specifically, but not with ΔRahU strain. Binding affinity of LDL/oxLDL with both wild P. aeruginosa strain and ΔRahU strain made no significant difference. Protease digestion did not influence the binding of ΔRahU strain to LDL/oxLDL. [Conclusion] RahU is one but not the unique ligand of LDL/oxLDL on P. aeruginosa.

Abstract:[Background] Intestinal flora performs a crucial role in human health and its imbalance may cause numerous pathological changes. Lactobacillus and Bifidobacterium are the main beneficial bacteria, which to some extent reflect the health status of the intestinal tract. However, the community structure of intestinal flora was different in different populations. [Objective] To analyze the diversity of lactic acid bacteria (LAB) and Bifidobacterium in the intestine of healthy Mongolians at the species level. [Methods] We studied the abundance and diversity of LAB and Bifidobacterium in 27 healthy Mongolians from Inner Mongolia Autonomous Region of China and Mongolia by using laboratory-designed LAB primers and Bifidobacterium-specific primers. This work also aimed to discuss the influence of gender, body mass index (BMI) and location on the quantities of these two bacterial groups, and to analyze the correlation between dominant strain. [Results] At the species level, a total of 68 LAB species and 11 Bifidobacterium species were detected in the intestine of 27 healthy Mongolians. Among them, eight LAB species were the most abundant species with relative abundances >1.0%, including Streptococcus salivarius (36.41%), Lactobacillus ruminis (17.94%), Lactobacillus delbrueckii (3.11%), Lactobacillus rogosae (2.23%), Streptococcus mitis (2.18%), Lactobacillus vaginalis (2.02%), Weissella confuse (1.54%) and Lactobacillus rhamnosus (1.09%). Meanwhile, Bifidobacterium adolescentis (39.88%), Bifidobacterium longum (27.15%), Bifidobacterium catenulatum (26.30%), Bifidobacterium bifidum (3.92%) and Bifidobacterium angulatum (1.71%) were the most abundant Bifidobacterium species with relative abundances>1.0%, and volunteers can be divided into two different groups (B. catenulatum group and B. adolescentis group) according to the composition of bifidobacteria by the cluster analysis. The results showed that the community structure of LAB and Bifidobacterium has no significantly correlation with gender, BMI value and region, and the community structure of LAB also no significantly correlation with BMI value, but there are significant differences in the relative abundances of several LAB species between men and women, between in Inner Mongolia and the Mongolian volunteers (P<0.05). This study also found significant correlations between dominant LAB and Bifidobacterium, and the correlation between different species was different and related to specific strains. [Conclusion] This is the first report on the use of PacBio SMRT sequencing technology to evaluate the diversity of lactic acid bacteria (LAB) and Bifidobacterium in the intestine of healthy Mongolians at the species level. Based on these data of intestinal flora, it is beneficial to achieve targeted regulation of intestinal flora and accurate medical treatment. In addition, our study could serve as good reference for other studies on the diversity of LAB and Bifidobacterium in the intestinal tract at the species level.

Abstract:[Background] With increasing numbers of drug resistant microorganisms and drained antibiotics sources, research on bacterial drug resistance mechanisms has risen to be a most urgent scientific topic. [Objective] To understand how endogenous cysteine affects ROS levels in Escherichia coli by promoting the Fenton reaction. [Methods] The effect of cysteine on the antibiotic resistance of E. coli was investigated by controlling the concentration of exogenous cystine and the concentration of antibiotics (gentamicin, ampicillin, norfloxacin) under poor sulfur conditions. [Results] Low concentrations of cysteine increased drug resistance of E. coli to all of the antibiotics used in this study. The regulation on Fur, CysB or SOS by influx of cystine was also confirmed using RNA-Seq. An analysis of sulfur efflux by LC-MS showed that excess cysteine was excluded immediately. At certain concentrations of antibiotics, the efflux pump AlaE exhibited favorable protection on cells. [Conclusion] Drug resistance of E. coli to gentamicin, ampicillin, norfloxacin, antibiotics with different mechanisms of action, was affected by endogenous cysteine levels. Our current work provides new theoretical information on bacterial drug-resistance mechanisms.

Abstract:[Background] The intestinal microflora of giant panda are abundant, and the population structure is related to the age of the host, living environment, seasonal changes and other factors, among which age is one of the important factors affecting the composition of intestinal microflora. [Objective] Study the diversity of Bacillus isolated from giant panda gut in different age, in order to explore the relationship between the diversity of Bacillus isolated from giant panda gut and age, which provide strain resources for the development of new probiotic. [Methods] Spread plate method was employed to isolate Bacillus. BOXA1R-PCR, 16S rRNA gene phylogenetic analysis and principal component analysis (PCA) were used to analyze the diversity of culturable Bacillus. Confrontation growth method and disk diffusion test were employed to test the antibacterial activity and antibiotic susceptibility of isolates. [Results] Total 90 strains belong to Bacillus genera were isolated. Based on the result of BOXA1R-PCR analysis, 41 representative strains were selected for further 16S rRNA gene sequencing. The result showed the isolates were affiliated to 6 species, including Bacillus subtilis, B. atrophaeus, B. velezensis, B. methylotrophicus, B. amyloliquefaciens, B. pumilus. The result of PCA showed the population composition of Bacillus in giant panda gut was influenced by age. All of representative isolates possessed CMCase ability, most of isolates exhibited antimicrobial activity to pathogens. All the 41 representative strains showed resistance to Penicillin G and susceptible to the other 9 antibiotics. [Conclusion] This study indicated that Bacillus isolated from giant panda gut was abundant and the distribution was influenced by age, which possessed great potential in developing probiotics and can be further explored.

Abstract:As the energy crisis and environmental problems are ongoing and getting worse, traditional chemical synthesis industry is being replaced by bacteria-based bio-industries for production of chemicals and fuels, in which, the most critical issue is developing proper genetic engineering tools to establish platform organisms for bioproduction. Derived from archaeal and prokaryotic immune systems to defend viral and plasmid invasions, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) are developed into a fast, easy, and robust genome editing tool in recent years and have potential to cater to the demands. Here, we briefly review the principle and advanced classification of CRISPR/Cas systems. The establishment, optimization and applications of CRISPR-based genome editing tools in the construction of prokaryotic microbial cell factories are introduced with the examples. Besides, some feasible solutions are proposed based on the discussion about the problems existed in CRISPR/Cas systems.

Abstract:Steroid compounds plays an important role in physiological and pharmaceutical effects and the market demand is very huge. The microbial transformation process of steroid compounds and their key steroids is gradually applied since its application offers a number of advantages over chemical synthesis as follows: regio-stereoselectivity, reducing the synthesis procedures, shortening the production cycle, improving yield, more eco-friendly process and so on. However, the mechanism of microbial catabolism of steroids is still to be further explored and determined. The essay comprehensively analyzes the structure types and main sources, physiological functions, microbial transformation and catabolic mechanisms of steroid compounds and focuses on the key enzymes and their molecular mechanisms of metabolic process of steroids, which can provide a reference for construction of engineering strains and the development of industrial production technology for microbial transformation of steroids.

Abstract:Pathogenic bacterial infection poses a serious threat to human health. Intestinal pathogenic bacteria that contain a Type III secretion system (T3SS) can use the T3SS to inject effectors into host cells and manipulate multiple signaling pathways, including apoptosis, autophagy and inflammatory responses pathways, in order to evade host defense system and enhance their infectivity and pathogenicity. Here, we have reviewed the latest research progress in the regulation of host NF-κB and MAPK pathways involved in inflammatory responses by the T3SS effectors of intestinal bacterial pathogens.

Abstract:The research progress and current status of scalp microbial diversity and anti-dandruff active ingredients were reviewed. The relationship between colonization and dandruff of Staphylococcus sp., Propionibacterium and Malassezia was introduced. The function and toxicity of the active ingredient zinc pyrithione and the compounding of anti-dandruff shampoo functional factors were prospected.

Abstract:Src and Abl family kinases are the important members of the nonreceptor tyrosine kinase (NRTK) family, which exist widely in various cell types, participate in different intracellular signaling pathways, and regulate a variety of cellular physiological processes. They play a vital role in maintaining the homeostatic function of the normal cells, tissues and organs. Studies have shown that Src and Abl family kinases are involved in the infection with multiple pathogenic microorganisms through a variety of mechanisms (e.g. interaction with the proline motif-PXXP of the pathogenic microorganism). Therefore, the study of mechanism of pathogenesis of microbial pathogen infection from the perspective of Src and Abl family kinases has become a hot research issue. In this paper, the structural characteristics of Src and Abl family kinases and the reports on the relationship between microbial pathogen infection and host cell kinases are systematically reviewed, which should provide references for the research of pathogenic mechanism, prevention and control and drug development of pathogenic microorganisms.

Abstract:With the rapid development of mobile network technology, its educational functions have been gradually explored, and show the prospect of good mobile learning applications. In this paper through building the learning resources base on “Zhejiang Online University APP” and “WeChat Public Platform”, we design blended teaching model based on mobile learning, and apply it to the teaching reform practice of Biochemistry courses. The results of classroom teaching practice and survey feedback show that applying mobile learning to mixed teaching is a popular teaching model with students and has a good teaching effect.

Abstract:Microbiology is a very important compulsory fundamental course in life science, medicine, pharmacy, agriculture, forestry, food, etc. for undergraduate teaching. Research in microbial diversity is becoming research hotspots in various fields and continues to achieve fruitful research results in recent years. However, majority of Microbiology textbooks for higher education lack the contents in research methods of microbial diversity and research progress in related field. These conditions limit students’ learning and mastery of knowledge and not feasible to expanding their horizons of learning. Here, based on comparative analysis of these knowledge contents of microbial diversity in the mainstream of Microbiology textbook used in China and abroad, we suggest to add them into the textbooks, which will help students to better understand the research progress.

Abstract:[Background] Escherichia coli O157:H7 is the main serotype of Enterohemorrhagic Escherichia coli causing outburst of foodborne disease. [Objective] Our goal is to prepare immunomagnetic beads (IMBS) which are high efficient, stable and broad-spectrum, and also to improve the detection rate of target bacteria from food samples by combining immunomagnetic separation and molecular detection technology like loop-mediated isothermal amplification (LAMP), PCR, etc. [Methods] We used MIX&GO as activating agent of carboxyl magnetic beads, and successfully prepared immunomagnetic beads coupled with commercial polyclonal antibody. Subsequently, its universality and specificity were evaluated. The preservation solution of immumomagnetic beads consisting of bovine serum albumin, casein, trehalose, polyvinyl pyrrolidone (PVP), ascorbic acid and ProClin300, has been optimized by orthogonal experimental design L18(37). Six detecting methods including IMBS-LAMP, IMBS-PCR, IMBS-biochemistry, LAMP, PCR and biochemistry respectively without IMBS were adopted to detect E. coli O157:H7 in 20 ground raw pork meat samples. [Results] The capture efficiency of prepared immunomagnetic beads is 81.5%±1.3% when applied in the sample matrix of PBS solution, but decreasing in complex food matrix. The optimal formula for the preservation solution of the immunomagnetic beads is bovine serum albumin 15.0 g/L, casein 10.0 g/L, trehalose 10.0 g/L, PVP 2.0 g/L, ascorbic acid 5.0 g/L, and ProClin300 2.5 g/L. The results showed that the most sensitive method was the combination of IMBS and LAMP; 9 positive samples were respectively detected by self-prepared IMBS-LAMP and commercial IMBS-LAMP. But there was an inconsistency in two groups of positive samples because of different antibody sources of self-prepared and commercial IMBS. [Conclusion] Compared to commercial immunomagnetic beads, the prepared beads have good specificity and broader spectrum of E. coli O157 strains. Our study also showed that the IMBS-LAMP scheme could effectively enhanced detection rate of target bacteria. The IMBS-LAMP technique could be considered a high sensitive detection method of application prospect.