Abstract:[Background] Tryptophan decarboxylases that catalyze tryptophan to tryptamine, have specificity to catalyze target substrate in the nature. A decarboxylase from marine Bacillus atrophaeus C89, involved in the biosynthesis of bacillamide C, is referred to as BaTDC. [Objective] We are aiming to characterize BaTDC and explore the substrate spectrum of BaTDC including halogenated tryptophans and hydroxytryptophan in order to provide new methods to produce novel and pharmaceutically vital tryptamine analogues. [Methods] A phylogenetic tree was constructed using protein sequences of several TDCs to understand the status of BaTDC in evolution. Its activity was assayed with various tryptophan derivatives and the products were detected by HPLC and UPLC-MS. [Results] Phylogenetic analysis revealed the similarity of BaTDC with that of the gut bacterium Ruminococcus gnavus. The optimum temperature and pH of the purified recombinant BaTDC enzyme was 40?45 °C and 8.0, respectively. BaTDC exhibited substrate promiscuity and catalytic efficiency with hydroxytryptophan and halogenated tryptophans including 4-fluorotryptophan, 5,6,7-chlorotryptophan and 4-bromotryptophan. [Conclusion] The study presents a comprehensive characterization of the BaTDC as a promising member of its enzyme family. BaTDC exhibits broad substrate tolerance to tryptophan derivatives, suggesting the potential of substrate-feeding approach in producing novel tryptamine analogs or complex secondary metabolite analogs through precursor-directed biosynthesis.

Abstract:[Background] Burkholderia sp. SJ98 utilizes para-nitrophenol or 2-chloro-4-nitrophenol as the sole carbon and energy source for its growth. The superoxide dismutase SodA from Haloferax sp. D1227 endows strain SJ98 with the ability to degrade para-nitrophenol with 500 mmol/L NaCl. However, it is unknown whether strain SJ98 containing sodA can degrade para-nitrophenol derivatives under conditions with high salt concentration. [Objective] We are aiming to study the salt tolerance of strain SJ98, and the degradation of para-nitrophenol and 2-chloro-4-nitrophenol by strain SJ98 containing sodA under normal and high salt concentrations. We also hope to detect the transcriptions of pnpA and the activities of the nitrophenols monooxygenase in the recombinants of strain SJ98. [Methods] Strain SJ98 and its recombinants were cultured in minimal medium (containing 400 to 800 mmol/L NaCl) or M9 medium (containing 0 and 500 mmol/L NaCl, respectively) supplemented with glucose, para-nitrophenol or 2-chloro-4-nitrophenol, respectively. The strains growth and substrates degradation were detected by UV spectrophotometer and high-performance liquid chromatography, respectively. The transcriptions of pnpA (nitrophenol monooxygenases encoding gene) induced by two nitrophenols were detected by quantitative real-time PCR. The activities of nitrophenols monooxygenase in the crude enzyme solutions were detected by UV spectrophotometer using two above substrates. [Results] The NaCl tolerance concentration of strain SJ98 was 600 mmol/L with glucose as the carbon source. The growth and para-nitrophenol degradation by strain SJ98[pCM-pnpR-PpnpA-sodA-rfp] were much better than those by the wild-type strain. With 500 mmol/L NaCl, strain SJ98[pBBR-sodA] still maintained the ability of degrading and growing on 2-chloro-4-nitrophenol, while strain SJ98[pBBR1MCS-2] completely lost these abilities; the activities of the nitrophenols monooxygenase against two above substrates in the crude enzyme solutions of strain SJ98[pBBR-sodA] were both about 1/3 of those in the wild strain. When two nitrophenols were used as inducers, the transcriptions of pnpA in strain SJ98[pBBR-sodA] were about 17–25 times higher than those in the wild-type strain, regardless of the presence of NaCl. However, when 500 mmol/L NaCl was added, the transcriptions of pnpA were partially inhibited. [Conclusion] This study provides a potential possibility for the application of superoxide dismutase from archaea to improve the ability of nitroaromatics degradation under normal and high salt concentrations by bacteria.

Abstract:[Background] Azo dyes and their degradation intermediates have certain environmental toxicity. The application of mixed flora to degrade azo dyes is environmentally friendly, whereas the presence of oxygen plays a vital role in the degradation process, which can promote or suppress the biodegradation of azo dyes. [Objective] The effect of oxygen on the azo dye decolorizing solutions were explored, and the influence of oxygen on the decolorization and degradation of azo dyes by mixed flora were investigated. [Methods] Through the decolorization and degradation of seven azo dyes by mixed flora DDMY1 under three culture conditions (aerobic, anaerobic and facultative), the response of their decolorizing solutions to oxygen were discussed. Meanwhile, the degradation products were detected by ultraviolet visible spectrophotometry (UV-vis) and Fourier transform infrared spectroscopy (FTIR). [Results] After 48 h reaction under facultative and anaerobic conditions, several azo dye decolorizing solutions were captured to have the remarkable color restoration after exposure to oxygen, such as Reactive Black 5 and Direct Black 38. The results of UV-Vis analysis showed that the phenomenon of color restoration was derived from the generation of the new substances produced after decolorizing solutions contact with oxygen. In addition, the outcome of FTIR analysis indicated that the mixed flora DDMY1 still had the decolorization and degradation effect on azo dyes despite of color restoration, but the decolorization was not complete. [Conclusion] Under facultative and anaerobic conditions, oxygen had obvious influence on partial azo dye decolorizing solutions, affecting the overall decolorization performance of mixed flora DDMY1 on azo dyes. The study provides a theoretical basis for further research on the complete biodegradation of azo dyes.

Abstract:[Background] Biogas and natural gas often contain a certain amount of hydrogen sulfide. Hydrogen sulfide is not only polluting the environment but is also harmful to humans. [Objective] In this study, a sulfur-oxidizing bacterium was screened from sodium alkali lake sediments in the Badain Jaran Desert using sodium thiosulfate as the sole energy source, and its sulfur oxidation properties were studied. [Methods] Strain BDL05 was identified by a variety of methods including Gram staining, colony morpholog, and 16S rRNA gene sequence analysis. [Results] Strain BDL05 was a Gram-negative bacterium, helical, belonging to the family of Ectothiorhodospiraceae and Thiomicrospira genus. The 16S rRNA gene sequence similarity of BDL05 to Thiomicrospira microaerophila ASL 8-2 reached 99.8%, so it was named as Thiomicrospira microscopila BDL05. The optimum pH for sodium thiosulfate was 9.3, and the optimum total sodium salt concentration was 0.8 mol/L. It had a strong ability to oxidize sodium sulfide. In the airlift reactor using sodium sulfide as the sulfur source, the yield of elemental sulfur was 94.7%, and the production rate was 3.0 mmol/(L·h). [Conclusion] This indicated that Thiomicrospira microaerophila BDL05 was a sulfur oxidizing bacteria which can be of a strong applicability in gas biological desulfurization.

Abstract:[Background] Naproxen is a widely used drug which is non-steroidal and anti-inflammatory. It has certainly negative impact on the environment while treating human diseases. What’s more, it maybe even endangers the human living environment. [Objective] The way of degrading naproxen pollutants through microorganisms is inexpensive and effective. [Methods] In this paper, naproxen was used as the sole carbon source to cultivate domesticated and highly effective naproxen-degrading bacteria; The high-throughput sequencing technology was used to analyze the microbial community changes of naproxen-degrading bacteria and to identify the types of naproxen-degrading microbial community; Analysis of degrading pathway of naproxen-degrading microbial community by GC-MS. [Results] Finally, a highly effective naproxen-degrading microbial community was mainly Rhodanobacter, and the optimal growth degradation condition of naproxen-degrading microbial community was determined to be 30 °C, pH 7.0, 150 r/min, 10% inoculation amount, with naproxen degrading rate reached 60.58%, and predicting the degradation pathway of naproxen-degrading microbial community. [Conclusion] Obtaining a high-efficiency naproxen- degrading microbial community, and clarifying the degradation mechanism and degradation pathway. The research not only enriches the types of microbial resources, but also lays the theoretical foundation for microbial engineering applications.

Abstract:[Background] The treatment of heavy metal pollution is imminent because heavy metal poses a major threat to the environment and human health. Biological treatment methods are preferred because of the advantages of low cost, good treatment effect and no secondary pollution. [Objective] From the 13 m deep water body of the Liaohe estuary, a strain possessing strong pigment-producing ability and high- concentration Cu2+ removal ability was screened by the purple non-sulfur bacteria enrichment medium. [Methods] Morphological, physiological and biochemical characteristics and molecular biological methods were used to identify the strain. The content of Cu2+ was determined by spectrophotometry with sodium diethyldithiocarbamate. [Results] The strain was identified as Rhodopseudomonas and named as Rhodopseudomonas sp. gh32. The optimal growth temperature and growth pH of strain gh32 were 30 °C and 7.0, respectively. The strain gh32 grew normally in the range of pH 5.0?10.0. The strain gh32 could grow normally in 3 mmol/L CuSO4 solution. The strain gh32 could utilize hydrogen sulfide and monosaccharides, such as glucose and mannose. The removal rate of Cu2+ was over 99% in strain gh32 within 24 h. The ability to treat Cu2+ was 1 331 g/g dry bacterial weight or 167.6 g/g wet bacterial weight. [Conclusion] The strain gh32 was highly resistant to Cu2+ and there was a high removal rate of Cu2+ by strain gh32. It will be a potential strain of treatment wastewater containing Cu2+. This study provided support for biological treatment of heavy metal wastewater.

Abstract:[Background] Pseudomonas protegens H78, a biocontrol strain isolated from the rape rhizosphere, can produce multiple broad-spectrum antibiotics such as pyoluteorin (Plt). Plt biosynthesis is completely inhibited by the rsmA/E dual mutation in Pseudomonas protegens H78. [Objective] The aim of this study is to screen the downstream regulatory factors for reactivating Plt biosynthesis in the H78ΔrsmA/E dual mutant by transposon mutagenesis. [Methods] the red fluorescent protein (RFP) gene was inserted downstream of the Plt biosynthetic gene pltL by homologous recombination to indicate the activation of Plt operon expression. the target gene was screened and located by the transposon-based random insertion mutation and the semi-random PCR. the gene function was further confirmed by the gene complement method. [Results] A mutant with high yield of Plt was screened from about 20 000 transposon insertion mutants of H78ΔrsmA/E, and its mutation site was determined to be within the hmgA gene. In turn, the hmgA complementation can inhibit Plt biosynthesis in the H78ΔrsmA/E strain. [Conclusion] In the H78ΔrsmA/E dual mutant of P. protegens, the hmgA gene shows strong inhibitory effect on Plt biosynthesis. The hmgA gene is a potential downstream regulatory gene of RsmA/E. This study lays a foundation for further elucidating the regulatory mechanism and network of Plt biosynthesis and improving the yield of Plt through genetic engineering.

Abstract:[Background] Pseudomonas PA1201 strain is a plant growth promoting rhizobacterium isolated from rice rhizosphere. Pyoluteorin (Plt) is one of the secondary metabolites produced by PA1201, which can effectively inhibit the growth of a variety of phytopathogenic fungi and bacteria. Under normal culture conditions, Plt yield is extremely low. [Objective] This study aimed to analyze the effects of different carbon sources for optimal Plt biosynthesis. [Methods] PA1201 was cultured in minimal medium (MM) supplemented with different carbon sources as the sole carbon source, or MM medium with different combinations of carbon sources. Plt was extracted at different time points after inoculation, and then quantitatively analyzed by high-performance liquid chromatography (HPLC). [Results] A HPLC-dependent method for quantitative analysis of Plt levels was established; MM medium supplemented with fructose and mannitol were found to be optimal for Plt production; no additive effect between fructose and mannitol was observed on Plt biosynthesis; In the MM medium containing mannitol or fructose, addition of glucose or succinic acid inhibited Plt biosynthesis. [Conclusion] Fructose and mannitol promote the synthesis of Plt in rice rhizobacterium Pseudomonas PA1201, laying a foundation for improving the biosynthesis efficiency of pyoluteorin and promoting the application of pyoluteorin.

Abstract:[Background] Lentinula edodes partitivirus 1 (LePV1) is a major mycovirus identified in L. edodes germplasm. Previous to this study, LePV1 isolates from the Chinese genetically-diverse L. edodes core collection were grouped into two distinct clades (subtype I and subtype II), with the majority in subtype I; The same LePV1 molecular isolate was found in L. edodes strains that were genetic-diverse and geographically far away. [Objective] To identify the effect of virus-transmission via basidiospore on the population of Lentinula edodes partitivirus 1. [Methods] We compared the virus-carrying rate of basidiospores between subtype I and subtype II, and analyzed the effect of hybridization (Mon-Mon and Di-Mon) on the population of LePV1. [Results] The virus-carrying rates of basidiospores was 70% in ZP51 and 100% in YS94 in subtype I, while it was 45% in ZP28 and 55% in YS5 in subtype II. Virus-transmission efficiency of basidiospore from strains in subtype I was higher than that of basidiospores from strains in subtype II. If one of the parental basidiospores carries LePV1, the obtained hybrid will carry LePV1 either in Mon-Mon crossing test or Di-Mon crossing test. [Conclusion] The different virus-transmission efficiency of basidiospore from strains in different subtypes, and plasmogamy duringhy bridization maybe play an important role in the formation of the population of LePV1. In addition, LePV1 was not spread from spores that carried virus to its incompatible monokaryons or dikaryons in Mon-Mon crossing test or Di-Mon crossing test. Additionally, the obtained hybrids could transmit LePV1 to the paring heterokaryons in the successful Di-Mon crossing test. This research presented here firstly provide experimental evidence and clues for us understanding the transmission characteristics and population formation of LePV1.

Abstract:[Background] Bipolaris papendorfii is the main fungal pathogen of Curvularia leaf spot, and harmful to agricultural production. At present, the use of bacteria to control B. papendorfii becomes research focus in this field. [Objective] To screen the strains with high antagonistic activity against B. papendorfii, and identify the antagonistic mechanism. [Methods] Twenty-two bacterial strains were isolated from the soil under the surface of corn field by the method of plate confrontation. One strain L-14 with high antagonistic activity to B. papendorfii was obtained by repeated screening. [Results] Strain L-14 with the highest antagonism was obtained by using the plate diffusion method. After morphological observation, 16S rRNA gene sequence analysis, physiological and biochemical characteristics detection, the strain was finally identified as Bacillus subtilis. Moreover, the strain is a wide-spectrum antagonistic biocontrol strain with inhibitory effects on Fusarium proliferatum, Fusarium graminearum, Exserohilum turcicum, Bipolaris papendorfii and Botrytis cinerea. Then, we studied the antibacterial mechanism of strain L-14 and obtained that the fermentation crude protein extracts had no effect on the morphology of the aerial hyphae of B. papendorfii but can cause the mycelial aberration, and had an inhibitory effect on spore germination. [Conclusion] The antagonistic bacterium B. subtilis obtained in this study has broad spectrum and high antagonistic activity in the control of plant diseases, and can effectively control Curvularia leaf spot.

Abstract:[Background] The potato common scab is a highly destructive disease caused by Streptomyces scabies, which leads to severe economic loss in the main producing areas of China. Due to the increasing disease risk and lack of efficient control means, identification of antagonistic microorganisms is becoming a hotspot in the relevant research field. [Objective] This study is aimed to screen antagonistic bacterial strains against Streptomyces scabies, ultimately providing candidates for developing applicable microbial agents. [Methods] The near-rhizosphere soil was collected from potato fields in Zhaotong, Yunnan, where severe scab disease raged, and strains with antagonistic effects were isolated. Morphological observation, physiological and biochemical analyses, and 16S rRNA gene sequence determination were performed to characterize the strains isolated. The stability and bacteriostatic function of their metabolites were further examined. [Results] A strain with high antagonistic capacity was obtained and named YN-2-2. The bacterial cells were rod-shaped and gram-positive with a size range of (2.51?4.09) μm×(1.09?1.68) μm. Its 16S rRNA gene sequence had 99.79% identity to Bacillus thuringiensis ATCC 10792T (ACNF01000156). The secondary metabolites of YN-2-2 showed good thermal stability, a wide pH tolerance between pH 3.0 and pH 13.0, and low sensitivity to proteinase K. The diameter of the largest inhibition zone against Streptomyces scabies was 22.8 mm. Pot experiments indicated that the disease index of common scab decreased significantly when potato plants were inoculated with 100 mL YN-2-2 culture with a final concentration of 1×107 CFU/mL, and the control effect was 36.11%. [Conclusion] Strain YN-2-2 was identified as Bacillus thuringiensis, it can be included into compound microbial agent against potato common scab.

Abstract:[Background] Flue-cured tobacco is an important economic crop in China. Intercropping affects the diversity of rhizospheric microorganisms and alleviates the production of flue-cured tobacco. Rhizospheric microorganisms can adapt to changes in the rhizosphere microenvironment, which promotes the growth of flue-cured tobacco. [Objective] We examined the diversity of the bacterial population in flue-cured tobacco’s rhizosphere soil under the intercropping models between tobacco K326 and five spice plants at different growth periods. In addition, we compared the variances in intercropping models in various growing periods from the perspective of biological diversity and subsequently screened the adaptability and functional characteristic of typical rhizosphere bacterium. [Methods] Soil samples from various growing periods with different intercropping patterns were collected. We designed the culturing method to isolate and sequence the 16S rRNA gene, and calculated the diversity index. Representative strains were selected to detect nicotine and Cr2+ tolerance as well as growth-promoting properties. [Results] A total of 707 bacterial strains distributed in 70 genera were isolated. The change of the Shannon index of bacteria in flue-cured tobacco’s rhizosphere soil at the stem elongation period was W (4.079 191 7)>2GAY (3.840 352 1)>2GDY (3.514 562 8)>2GCKY (3.497 703 4)>2GBY (3.447 478 9)>2GCY (3.253 409 2)>2GEY (3.241 103 4). The change of Shannon index of bacteria in flue-cured tobaccos’ rhizosphere soil at the leaf ripening period was 3GAY (3.688 981 7)>3GCKY (3.442 125 1)>3GBY (3.353 155 8)>3GDY (3.349 171 9)>3GEY (3.306 294 5)>3GCY (3.305 582 2). The change of Shannon index of bacteria in flue-cured tobacco’s rhizosphere soil at the harvesting period was 5GEY (2.857 102 8)>5GBY (2.458 311 3)>5GAY (2.271 868 5)>5GCKY (2.210 253 6)>5GCY (2.079 441 5)>5GDY (0.693 147 2). The genera Arthrobacter, Bacillus, Brevibacillus, Brevendimononas, Microbacterium, Nocardioides, Pseudomonas, Rhodococcus, Streptomyces and Terrabacter were detected at all the three growth periods. Majority of the representative strains exhibit positive activities for nicotine tolerance, Cr2+ tolerance, nitrogen fixation, siderophore production, phosphorus solubilization and starch hydrolysis. [Conclusion] Rhizosphere soil contained abundant bacterial resources under the intercropping mode between five spice plants and the flue-cured tobacco K326. Among the dominant bacterial groups in flue-cured tobacco’s rhizosphere soil, the genera Bacillus, Pseudomonas and Streptomyces have potential applicability in nicotine tolerance, Cr2+ tolerance and growth-promoting effects. The intercropping can change the diversity of bacteria in rhizosphere soil of flue-cured tobacco. In the tobacco leaf ripening period, the intercropping of five spice plants and flue-cured tobacco significantly increased the diversity of cultivable bacteria in the rhizosphere soil. The intercropping types of Monarda didyma, Cymbopogon citratus and Pelargonium graveolens own advantages in relieving continuous cropping of flue-cured tobacco.

Abstract:[Background] Root rot is highly susceptible to the cultivated Aconitum carmichaelii Debx. in Jiangyou area of Sichuan, China. The plants wither and die after being infected by root rot, which ultimately affects the yield of medicinal materials. The prevention and treatment of the root rot are difficult due to complex pathogenic factors and the diversity of pathogenic fungi. [Objective] To identify the pathogens of root rot on A. carmichaelii Debx. cultivated in Jiangyou area, and to provide theoretical basis for diagnosis and prevention of the disease. [Methods] Pathogen isolation was carried out by means of diseased tissue isolation method. Koch’s rule was used to prove the pathogenicity of fungal strain isolated from A. carmichaelii. The pathogens were identified based on their morphological characteristics and rDNA-ITS sequence. [Results] Forty-seven fungi isolates were isolated from the root rot of A. carmichaelii. Strains GF3-3, GF3-6 and GF6-1 were the causal pathogen fungi of the root rot of A. carmichaelii. The pathogen strains GF3-3, GF3-6 and GF6-1 were identified as Fusarium solani, F. oxysporum and F. proliferatum via morphological characteristics and rDNA-ITS sequence. [Conclusion] Fusarium solani, F. oxysporum and F. proliferatum were the principal pathogenic fungi causing root rot disease of A. carmichaelii. This is the first report of root rot of A. carmichaelii caused by F. proliferatum.

Abstract:[Background] Robinia pseudoacacia ‘Hongsen’ is a fast-growing deciduous tree of Robinia in Leguminosae. Endophytic diazotrophic bacteria of Robinia pseudoacacia ‘Hongsen’ is being researched little nowadays. [Objective] We studied the diversity and growth promoting characteristics of the culturable endophytic diazotrophic bacteria isolated from Robinia pseudoacacia ‘Hongsen’. [Methods] Strains were isolated and purified from surface sterilized roots, stems, leaves and root nodules of Robinia pseudoacacia ‘Hongsen’, the isolates were clustered by using IS-PCR finger-printing, nitrogenase activity of the representative strain of each group was measured, we also conducted phylogenetic analysis of 16S rRNA gene, physiological and biochemical tests and growth promoting experiments of the representative strains. [Results] A total of 56 strains were isolated from Robinia pseudoacacia ‘Hongsen’, belonging to ten groups: Paenibacillus sabinae, Klebsiella michiganens, Kosakonia radicincitans, Kosakonia pseudosacchari, Mesorhizobium erdmanii, Mesorhizobium huakuii, Mesorhizobium silamurunense, Pseudomonas geniculate, Burkholderia territorii and Devosia riboflavina, in which 5 groups were associative nitrogen-fixing bacteria, 3 groups were symbiotic nitrogen-fixing bacteria, 2 groups had not the nitrogen-fixing ability, which showed the rich diversity of endophytic diazotrophic bacteria of Robinia pseudoacacia ‘Hongsen’. Growth promoting experiments showed that 7 groups had the function of phosphorus solubility, 6 potassium solubility, 6 auxin secretion, 6 siderophore production, and 2 proteases production. [Conclusion] Endophytic diazotrophic bacteria isolated from Robinia pseudoacacia ‘Hongsen’ have abundant genetic diversity and growth promoting characteristics, which have potential value in exploitation and utilization of agricultural microbial fertilizer.

Abstract:[Background] Rotation was known as one of the effective methods in continuous cropping obstacle prevention. Banana-pineapple rotation has been confirmed to be a very effective method in banana Fusarium wilt disease control. [Objective] In order to evaluate the effectiveness of prevention to banana Fusarium wilt disease induced by different pineapple varieties, five treatments in banana orchard soil with high incidence of disease were set as follows: fallow (CK), banana planted (B), “bali pineapple” planted (B_BP), “gold pineapple” planted (B_GP) and “tai nong 17 pineapple” planted (B_PP). The amount of cultivated Fusarium oxysporum, total fungi, bacteria, actinobacteria and soil properties were investigated to analyze the characteristics of chemical properties and microbial distribution in different treatments. [Methods] Pot experiment and culturable microorganisms’ analysis were used to study the changes of soil properties and culturable microorganisms of different pineapple varieties planted in banana orchard with high disease. [Results] Compared with fallow (CK), amount of Fusarium oxysporum in treatment B was significantly increased, while in B_GP and B_PP treatments, it was significantly reduced. Besides, the number of culturable bacteria and actinobacteria were significantly increased in B_GP and B_PP treatments. Content of available phosphorus, numbers of bacteria and actinobacteria were negatively correlated with culturable Fusarium oxysporum, respectively. while soil pH and the number of culturable fungi were positively correlated with culturable Fusarium oxysporum. Results of principal co-ordinate analysis (PCoA) and multiple regression tree (MRT) showed that soil fertility quality of B_GP and B_PP treatments were similar, while, they were far different from the other three treatments. [Conclusion] Our finding provides evidence that planting “tai nong 17” and “gold pineapple” were more effective than “bali pineapple” in control of banana Fusarium wilt for their high-efficiency ability to improve the soil properties and the culturable microorganisms.

Abstract:[Background] orf3 is located between the s and e genes of porcine epidemic diarrhea virus (PEDV) genome. It is the only accessory gene found in PEDV and encodes the ORF3 protein. Our earlier study showed that the ORF3 protein might has an effect on cell apoptosis induced by PEDV infection. [Objective] To study the virulence mechanism of ORF3 protein in the process of PEDV infection and replication. [Methods] Vero cells were infected with 3 different PEDVs, namely rDR13att-?ORF3 (with orf3 deleted from the genome), DR13-ORF3att (with a N-terminal 92 aa truncated orf3) and rDR13att-ORF3wt (with full length orf3). We observed cell pathogenic effect (CPE). Then different methods such as live cell imager, flow cytometry, TUNEL assay were used for apoptosis analysis of the cells at different time points of infection. Analysis of major apoptosis-related protein such as the cleaved Caspase-3 was also carried out by Western blotting in PEDV-infected cells. Finally, transcriptome sequencing was used to study the differential expression genes within the cells infected by the different viruses. Transcriptome results were further verified by real-time PCR. [Results] rDR13att-?ORF3 induced more CPEs in the infected cells than the other two viruses. Dynamic observation results of live cell imager showed all the three viruses induced obvious apoptosis in the infected cells. However, higher apoptotic level was detected in the cells infected with rDR13att-?ORF3 than the cells infected by the other two viruses (P<0.05). The flow cytometry also checked the apoptotic difference with the cells infected with the viruses: higher proportion of apoptotic cells among the cells infected with rDR13att-?ORF3. The result was further confirmed by terminal deoxynucleotidyl transferase (TDT)-mediated dUTP nick end labeling (TUNEL) staining. There are more TUNEL staining cells among the cells infected with rDR13att-?ORF3 than the cells infected by the other two viruses. The result of Western blotting showed rDR13att-ORF3wt can inhibit Caspase-3 activation in the infected cells. Transcriptome analysis found there was a significantly higher-level heat shock 70 kD protein 1B (HSP70) transcription in cells infected with rDR13att-ORF3wt than in the cells infected with rDR13att-?ORF3. Real-time PCR showed the expression of HSP70 in cells infected by rDR13att-ORF3wt was higher than that of rDR13att-?ORF3. [Conclusion] ORF3 protein inhibited cellular apoptosis induced by porcine epidemic diarrhea virus infection. The effect may be through inhibition of Caspase-3 activation (cleavage) or promotion of HSP70 expression.

Abstract:[Background] Haemophilus parasuis (HPS) is the pathogen of Gl?sser?s disease. Currently in the prevention and control of the disease, due to antibiotic resistance and the lack of cross-immunity protection of inactivated vaccines, new methods are urgently needed to solve this problem. [Objective] The aim of this study was to explore the cross-immunoprotective properties of different serotype HPS transferrin-binding protein A (TbpA) in guinea pigs for further research in piglets. [Methods] Recombinant TbpA of HPS serotypes 4, 13 and 14 were immunized at 0.1 mg with 20-day interval and then detected the serum antibody (IgG) and cytokines (IL-2, IL-5, IL-8, IFN-γ, MCP-1 and TNF-α) by ELISA. HPS-BZ, HPS-LJ3 and HPS-SZ strains were dosed at 5 LD50 by intraperitoneal injection. Then, the pathological changes and immune protection rate were examined. [Results] Recombinant TbpA of HPS serotypes 4, 13 and 14 could induce high levels of antibody (IgG) and cytokines, and provide cross-immunity protection to guinea pigs. Among them, the HPS serotype 13 TbpA immunization group had the highest cross-immunity protection rate, both serotype 4 and 14 HPS were 50.0%, and the highest immune protection rate against the same serotype HPS was 83.3%. The pathological examination showed that compared with HPS serotype 4 and 14 TbpA immunization group, the difference between the pathological changes of HPS serotype 13 TbpA immunization group and the challenge control group was more significant. [Conclusion] Recombinant TbpA of HPS serotypes 4, 13 and 14 can induce the humoral and cellular immune responses in guinea pigs, among which HPS serotype 13 TbpA has the strongest cross-immunity protection and can be used as a new vaccine candidate antigen.

Abstract:[Background] D-1,2,4-butanetriol (BT) is an important four-carbon polyol with a wide range of applications in industries. The four-step biochemical reaction using xylose as a substrate is currently the most efficient BT biosynthetic route. However, the Escherichia coli host has serious carbon catabolite repression which limits the growth and BT synthesis of xylose and glucose sugar mixture. Klebsiella pneumoniae has the advantages of faster growth rate, better utilization of xylose and glucose sugar mixture. [Objective] Establishing BT synthesis pathway using xylose as a substrate in K. pneumonia, which has a weak carbon catabolite repression, to improve BT production of sugar mixture. [Methods] The heterologous pathway of BT biosynthesis was constructed in K. pneumoniae ZG25 by co-overexpressing the xylose dehydrogenase gene (xdh) from Clostridium crescenti, 2-ketoisovalerate decarboxylase gene (kivD) from Lactococcus lactis, and xylose dehydratase gene (yjhG) from E. coli W3110, obtaining recombinant strain K. pneumoniae ZG25-BT. After optimization of the medium and culture conditions and deletion of xylA, significant increase in BT production was achieved. [Results] The optimal procedure for BT production in recombinant K. pneumoniae ZG25-BT strain without the xylA was achieved in 1.5-fold LB medium under the conditions of 30.0 g/L xylose, 10.0 g/L glucose, 37 °C, 200 r/min rotation speed, adding 10.0 g/L CaCO3 to control pH and 1% inoculation amount followed by 2 h of induction with the addition of IPTG, which made BT titer, molar conversion and yield up to 4.52 g/L, 0.21 mol/mol and 15%, respectively, representing a 150%, 62% and 67% increase compared with that of unoptimizable condition, respectively. [Conclusion] A fermentative process of BT production in K. pneumoniae ZG25 is achieved. Optimization of the culture conditions and medium and the knockout of xylA are applied to improve BT production. Furthermore, this work provides a useful host for the improved BT production through metabolic engineering.

Abstract:[Background] Bacillus amyloliquefaciens BLCC1-0238 supplementation has been reported to effectively improve production performance and egg quality of laying hens in our previous study, the underlying mechanism, however, remains poorly understood. [Objective] The current study was conducted to examine the potential mechanism of B. amyloliquefaciens BLCC1-0238 to increase production performance of laying hens by evaluating cecal microflora and gene expression in ileum mucosa. [Methods] High-throughput sequencing technology was used to compare the differences of cecal microflora composition and gene transcription level of ileum mucosa between the basal diet group (group C) and B. amyloliquefaciens BLCC1-0238 supplementation group (0.06%, 2.0×1010 cfu/g, group T). [Results] B. amyloliquefaciens BLCC1-0238 supplementation can produce higher indexes of Chao1 and Shannon, which demonstrated increased diversity of cecal microflora. At the phylum level, the ratio of Firmicutes/Bacteroidetes was significantly increased, while relative abundances of Fusobacteria and Proteobacteria were reduced, and at the genus level, the relative abundances of Phascolarctobacterium, Lactobacillus, Ruminococcaceae UCG-014, Anaerotruncus, Ruminiclostridium 9, and Christensenellaceae_R-7_ group were all elevated, which indicated that B. amyloliquefaciens BLCC1-0238 supplementation altered the cecal microflora composition from different levels. With respect to gene expression change in ileum mucosa, 356 differentially expressed genes were identified, among which 199 genes were up-regulated and 157 genes were down-regulated. Bio-informatics analysis found that these up-regulated genes were involved in numerous signaling pathways associated with nutrition metabolism, such as glycine, serine and threonine metabolism, starch and sucrose metabolism, and galactose metabolism, which may accelerate nutrition absorption of laying hens. [Conclusion] B. amyloliquefaciens BLCC1-0238 supplementation can effectively improve laying hen production performance via increasing the diversity of cecal microflora and promoting nutrition absorption of laying hens, providing solid evidence for its further practical application.

Abstract:[Background] Grifola frondosa showed degenerated characteristics due to long-term subculture. [Objective] To restore the biological activity and characteristics of G. frondosa through rejuvenation method, and to acquire strains possessing improved vitality and genetic stability through mutagenesis by high efficient apparatus. [Methods] Hyphae tip isolation method was utilized for rejuvenation of the strains cultivated in PDA enriched medium and PDA-chestnut shell medium respectively. Rejuvenated strain P-2 regained its biological activity and properties. To further improve its production performance, P-2 was subjected to ARTP mutagenesis, and a mutant b-35 with promoted performance and high genetic stability was obtained finally. [Results] The dry weight of mycelium and polysaccharide content of the strain P-2 were 1.18% and 19.01%, and the growth rates were 35.17% and 35.11% respectively compared with that of the original strain. Cultivation of P-2 in fermentation tank indicated that the fermentation cycle was shortened to 32 h from 48 h, showing significant improvement of fermentation activity and efficiency. Mycelium dry weight and polysaccharide content of the mutant b-35 reached 1.56% and 25.07%, 40.15% and 39.33% higher than that of P-2. [Conclusion] ARTP mutagenesis method was proved to be an important way in breeding of high yield Grifola frondosa because it is easily operated, pollution-free and efficient.

Abstract:Perfluorinated compounds (PFCs) is a class of hydrocarbons (and derivatives) in which hydrogens are replaced by fluorine atoms. Perfluorooctane sulfonate (PFOS) is a representative PFCs. It has many aspects of toxicity for living organisms. Research shows that PFOS almost exists everywhere and makes pollution, and the degradation of PFOS becomes an urgent problem. However, PFOS has high stability to be degraded. In particular, studies of successful PFOS biodegradation are limited. Here, we introduce the developing status and highlight challenges of PFOS degradation. In addition, we suggest potential pathways for PFOS biodegradation.

Abstract:Straw degradation by microorganisms has become a research hotspot in the world with the rapidly development in renewable biomass energy. This paper aimed to analyze the trend of straw degradation by microorganisms, and provide information for the researchers in the field of straw degradation and promote the development of new biomass energy in China. This paper analyzed the number of papers, top countries, journals, institutes and authors in the field of straw degradation by microorganisms using the Web of Science database and the HisCite and VOSviewer software. The worldwide researches on straw degradation by microorganisms mainly focused on applied microorganisms, agriculture and biotechnology. The researches on straw degradation by microorganisms had been increasing year by year since the new century, among which the United States, China, Germany and Japan were in international leading level. The United States ranked first in terms of volume and influence. Although China ranked second in terms of volume and has a certain foundation, but its influence is still low. So it is necessary to strengthen the high-level research to promote the overall improvement of straw degradation by microorganisms in China.

Abstract:Viral metagenomics is a new approach to genomics research. With the rapid development of high-throughput sequencing technology, viral genomes from the environment can be quickly discovered, identified and described based on composition characteristics. In the past decade, many novel viruses have been discovered by viral metagenomics, enhancing the understanding of virus composition, distribution and diversity in different environments. Therefore, viral metagenomics has become an effective tool for clearly depicting the viral map in various special environments and understanding the distribution dynamics of viruses in nature. This article mainly summarizes the concept of virus metagenome, sample preparation and extraction method of virus total genome, sequencing technology, the application and development prospects of virus metagenome.

Abstract:Microalgae are promising biological resource, which will play important role in environmental protection, wastewater treatment and clean energy production. However, the high-cost of the harvesting process limits its large-scale application. Thus, it is quite important to find economical, environment-friendly, and efficient microalgae harvest technologies. This paper reviewed the advantage and disadvantage of different microalgae harvesting methods, including centrifugation, sedimentation, filtration, flotation, and flocculation technology. This paper focus on research progress about flocculation technology. The main aim is hoping to provide some theorical and technological support for economic, environment-friendly, efficient harvesting technologies to promote the microalgae industry.

Abstract:The microbiota-gut-brain axis has received increasing attention due to its possible biological basis in regulating mental illness. The gut microbiota is closely related to neuropsychiatric diseases such as depression. It can affect the development of depression through interaction with the “gut-brain axis”. However, the mechanism between gut microbiota and depression is not yet very clear. In-depth studies of the realtionship between microbiota-gut-brain axis and depression help us to recognize and understand the depression from another perspective. Regulating the composition of gut microbiota to treat and prevent neuropsychiatric diseases such as depression is one of the future research directions, which also provides new ideas for exploring the mechanism of Chinese medicine treatment and prevention of depression.

Abstract:Corona virus disease 2019 (COVID-19) has been postponed since the outbreak of the new coronavirus pneumonia. However, the suspension of classes has ceased to teach and suspend classes. Teachers of all schools use various network resources to carry out online teaching, which also brings opportunities for further exploring the new teaching mode of “Internet+education”. Taking the application of the cloud textbook of Immunology and Pathogenic Biology as an example, this paper discusses how to create a reasonable learning situation by using the mobile interactive digital textbook, so as to realize the interactive learning and improve the teaching effect of online learning.

Abstract:Carrying out ideological and political education in Microbiology course was conducive to improving the ideological and moral cultivation and comprehensive quality of students of related majors. It was the specific requirement of the fundamental mission of fostering morality and cultivating talents in current college education, and an important embodiment of the routinization and normalization of ideological and political education. One of main teaching methods of ideological and political education in Microbiology course was to improve students’ ideological and moral level by guiding them to understand the ideological connotation of some famous scholars and their deeds in the development of microbiology. This method had achieved ideal teaching effect in practice. However, there were some drawbacks of this method, such as insufficient design and lack of fashionability. In view of these problems, this paper proposed a problem-based learning design of ideological and political education in Microbiology course based on the case about the outbreak of coronavirus disease 2019 (COVID-19). The design was intended to use some advanced deeds or bad phenomena which happened in the outbreak of COVID-19 by refining and relating them with knowledge of microbiology and classic case in the history to carry out the problem-based learning. The COVID-19 outbreak was an important event in students’ personal experiences. Therefore, using the COVID-19 case could greatly improve students’ sense of participation and arouse their resonance. It would be a beneficial supplement to classic case used in microbiology ideological and political education. It could lead students to explore the ideological and ethical significance of each case and internalize it into their own value belief while learning professional knowledge. It would be helpful for students in fostering correct world outlook, views on life and values. The design could be a good way to achieve the goal, combine ideological education with scientific knowledge learning, of ideological and political education in Microbiology course.

Abstract:Animal Microbiology is an important basic course for animal husbandry and veterinary medicine in higher vocational school. It is theoretical, technical and practical. Its teaching effect has a significant impact on the students’ follow-up professional learning and practical skills. Starting from the requirements that the course teaching should consider the training of modern professional and technical personnel, this article discusses the necessity of reforming and practicing the Animal Microbiology course. Besides, this article suggests adjusting the teaching hours, to optimize the teaching content, to innovate the teaching methods and the comprehensive use of multiple teaching methods such as multimedia, flipped classroom, integration of theory and practice, etc. Finally, an efficient classroom of Animal Microbiology courses oriented by professional skills is built, and then a practical approach is proposed to train high quality and application-oriented with professional theoretical knowledge and certain practical operational skills.

Abstract:[Background] Ribulose-1,5-bisphosphate caboxylase/oxygenase (Rubisco) is a key enzyme and the rate-limiting enzyme of CO2 fixation in the Calvin cycle, and widespread in plants, algae and other autotrophic microorganisms, playing an important role in biomass synthesis and the global carbon cycle. In view of the importance and the extremely low carbon-fixation activity of Rubisco, it is of great significance to study the selection evolution of Rubisco. [Objective] This study aims to construct a selection system suitable for screening efficient Rubisco carboxylation. [Methods] The reason of the absence of selection pressure on Rubisco carboxylation in the existing system was analyzed, then the new selection system applicable for high efficiency Rubisco carboxylation was designed and constructed. The lactate-producing strain BWLac was used as the host, the growth of Rubisco expression strains with different carboxylation activities were compared under the anaerobic culture containing 5% CO2, HPLC and LCMS were used to detect total lactic acid production and the yield of lactic acid from fixed CO2 for the evaluation of new constructed selection system. [Results] The design principle was considered to base on the pull-down force generated by the terminal metabolite lactic acid and the balance of the residual NADH generated by glycerol metabolism. These two parts could enhance the metabolic flux of the carbon-fixing branch of Prk and Rubisco, which could strengthen the inhibition of the toxicity of RuBP, coupling cell growth effectively with Rubisco activity to construct a high-throughput screening approach. In the newly constructed selection system, the Rubisco inactivated mutant BWLac/197 could not grow, while the growth of Rubisco and Prk double inactivated mutant BWLac/197-2021 was not affected with the colony size was about 1.58 cm. RBC1 and 7002, which carboxylation activities were detected to be more than 2 times different, the cell growth was inhibited to varying degrees under the screening conditions, and the colony size was 1.06 cm and 0.65 cm, respectively. The lactic acid production and glycerol consumption were also tested to evaluate the effectiveness of the selection system. RBC1 consumed 1.39 g/L of glycerol, produced 2.82 g/L of lactic acid, and labeled lactic acid content of 18.05 μmol/L, which was 1.3?1.6 times higher than the corresponding test result of 7002. The detection results were consistent with the design principle of the selection system, the higher the lactic acid production, the more glycerol consumption, the higher the Rubisco carboxylation, the better the cells grow. [Conclusion] An efficient selection system for Rubisco carboxylation was successfully designed and constructed to provide an effective high-throughput screening method for the evolution or exploration of Rubisco with higher carboxylation activity.

Abstract:[Background] Sphingobium is a kind of bacteria which can effectively degrade polycyclic aromatic hydrocarbons (PAHs) represented by phenanthrene. This characteristic makes sphingobium have broad application prospects in the field of environmental pollution control and biotechnology. [Objective] The aim of this study is to optimize the quantification method in order to acquire the complete differentially expressed proteins of Sphingobium yanoikuyae SJTF8 under phenanthrene stress. [Methods] Data dependent acquisition (DDA) and data-independent acquisition (DIA) proteomics analysis methods were used in this experiment. [Results] These two methods had commonly quantified 580 differentially expressed proteins, which played important roles in cell metabolism, transport and regulation. [Conclusion] DIA has a significant advantage in the quantification of differentially expressed proteins, which is helpful for the discovery of low abundance regulatory proteins induced by phenanthrene stress.

Abstract:[Background] Calcein UltraGreen? AM is a novel fluorescent dye used to label and monitor living cells. [Objective] A rapid detection method of viable bacteria was developed based on the characteristics of the Calcein UltraGreen? AM. This fluorogenic probe gives strong and stable fluorescence signal in living cells, its fluorescence intensity is proportional to the number of living cells. [Methods] The cells of bacteria, especially the viable cells, were stained with Calcein UltraGreen? AM, and then, its fluorescence intensity was measured. The standard curves of the fluorescence intensity value-viable bacteria number were established by simultaneously number counting of viable bacteria using the plate method. [Results] The optimum pH for staining viable cells of bacterial was 8.0. Under a specific staining temperature, the total number of viable bacteria can be readily detected within 20?30 min. The standard curves of the total number of both Gram-negative and Gram-positive bacteria, including Pseudomonas aeruginosa NY3, E. coli, Bacillus, Rhodococcus erythropolis FF, Staphylococcus aureus, and Bacillus subtilis, vs relative fluorescence intensity was established. A good linear relationship (R2>0.99) for the six bacteria were obtained when the OD600 of the bacterial suspensions were in the range of 0.01?0.30. [Conclusion] When the concentration of the sample suspension was ajusted to 105?109 CFU/mL, a rapid, convenient, accurate, reproducible and stable detection method was developed with good recovery and accuracy. This established method can be reaily used to measure the living bacteria in the fields of microbial experiments, solid bacteria fermentation, food hygiene and safety, and environmental mornitoring.

Abstract:[Background] Norovirus is one of the most important pathogens causing acute gastroenteritis, and it is rich in genetic diversity. [Objective] This study intends to establish a simple and rapid method for colloidal gold immunochromatographic assay of Norovirus epidemic strains. [Methods] Monoclonal antibody 1B10 against Norovirus capsid protein labeled with colloidal gold. Monoclonal antibody 1D6 against Norovirus capsid protein and goat anti-mouse antibody was blotted on nitrocellulose membrane as test line and control line, respectively. Optimize the assembly conditions of the test strip, including the labeling pH, the concentration of the gold-labeled antibody and the concentration of the test line and control line. Evaluate the performance of the method, including sensitivity test, specificity test, shelf life test and coincidence rate. Finally, the method was applied to the detection of clinical samples to evaluate its application effect. [Results] The limit of detection was 5.9×105 copies/μL. This method did not cross-react with common diarrhea viruses such as Rotavirus, Astrovirus, Adenovirus, and Enterovirus. This method has a good repeatability. The test strip can be stored for at least one year at room temperature. A total of 24 samples were detected by the method. The positive coincidence rate between the test strip and the real-time fluorescence quantitative RT-PCR method was about 83% (15/18). Epidemic strains GII.2, GII.3, GII.4, and GII.17 were successfully detected by the method. [Conclusion] The established colloidal gold test strip method has good specificity and stability, and can be used for the detection of Norovirus epidemic strains and large-scale epidemiological investigation.