Abstract:[Background] Thermophilic archaea Candidatus Syntrophoarchaeum was found to coexist with sulfate-reducing bacteria and oxidize n-butane by the reverse methanogenesis pathway. However, in this process, the enzyme responsible for catalyzing the oxidation of butyl-CoM has not been determined yet. [Objective] Using molecular dynamics simulation to prove that the enzymes encoded by mtaA genes found in Ca. Syntrophoarchaeum can specifically catalyze the transfer of butyl in butyl-CoM, and they are not methyltransferases. [Methods] Using the crystal structure of Methanosarcina mazei coenzyme M methyltransferase MtaA (PDB ID:4ay8) as a template, homology modeling of MtaA_1 (GenBank ID:OFV65993.1) and MtaA_2 (GenBank ID:OFV65678.1) was performed. Molecular docking was used to obtain the structure when they were combined with CH₃-CoM and C₄H₉-CoM respectively, and AMBER18 was used for molecular dynamics simulation. [Results] When combined with C₄H₉-CoM, MtaA_1 and MtaA_2 exhibited a TIM-barrel-like three-dimensional structure similar to the fold of 4ay8. However, there are differences in the shape of the active site, the distance between Zn²⁺ and the substrate, and the coordination of amino acid residues around the active site. These differences may be the reason why the enzymes encoded by mtaA genes found in Ca. Syntrophoarchaeum catalyze the oxidation of butyl-CoM. The overall structure of MtaA_2 is more similar to 4ay8, and the coordination of residues around the active site is complete, suggesting that MtaA_2 is more likely to be active. When MtaA_1 and MtaA_2 bind to CH₃-CoM respectively, their overall structures are unrealistic, and the coordinated Zn²⁺ is too far away from the substrate, indicating that CH₃-CoM is almost impossible to bind to the enzyme. [Conclusion] MtaA_1 and MtaA_2 of Ca. Syntrophoarchaeum are likely to be specific butyl-transferases rather than catalyzing the transfer of methyl groups, and MtaA_2 is more likely to be active.

Abstract:[Background] Alkaline protease is the enzyme with the largest proportion among industrial enzymes and is widely used in cleaning, food, medical and other industries. Recent studies have found that alkaline protease has great potential in the production of biologically active peptides, which will further broaden its application in the field of health food. [Objective] In this study, Bacillus subtilis was used to heterologously express the alkaline protease SubC derived from Bacillus licheniformis.[Methods] By screening three kinds of Bacillus subtilis host strains (B. subtilis 1A751, MA07 and MA08) and six kinds of signal peptides (AmyE, AprE, NprE, Pel, YddT and YoqM), as well as optimizing the inducer concentration, fermentation medium and fermentation duration, we finally obtained the optimal recombinant strain MA08-AmyE-subC_opt. [Results] The extracellular enzyme activity of the recombinant strain MA08-AmyE-subC_opt was 3.33×10³ AU/mL. Secretory expression level is four times more than that of intracellular soluble protein expression. Compared with the control group WT, the enzyme activity increased by 73.4%.[Conclusion] The heterologous alkaline protease SubC was successfully expressed in Bacillus subtilis, which provided a theoretical basis for the expression of alkaline protease SubC and its industrial application in the field of health food.

Abstract:[Background] Phytic acid is an organophosphorus compound which are widespread in plant tissues. It can chelate nutrient elements such as metal ions and protein in plant tissues, making them unable to be absorbed and utilized by herbivorous monogastric animals. The phytase can effectively catalyze the hydrolysis of phytic acid. [Objective] In order to promote the research and industrial application of Debaryomyces castellii phytase, we constructed a recombinant strain that heterologously expressed Debaryomyces castellii phytase in Pichia pastoris. [Methods] The phytase gene from Debaryomyces castellii was optimized and transformed into Pichia pastoris GS115. The high expression strain was obtained by screening copy number, knocking out proteases, and co-expressing molecular chaperones and transporters. [Results] The enzyme activity in the fermentation supernatant of the recombinant strain GS115/DCphy(ΔPep4)(BFR2) is 7 times that of the low-copy strain. [Conclusion] The results can provide some guidance for the heterologous expression and potential industrial application of Debaryomyces castellii phytase.

Abstract:[Background] Aspergillus fumigatus α-1,2-mannosidase MsdS is an enzyme that cleaves N-linked Man₈GlcNAc₂ in Golgi apparatus to produce Man₆GlcNAc₂ on mature secreted glycoproteins. MsdS has been shown to play a significant role in morphogenesis, cell wall synthesis and protein secretion in A. fumi gatus. Unlike A. fumigatus, Trichoderma reesei produces Man₈GlcNAc₂ on its mature secreted glycoproteins and grows normally. These observations suggest a species-specific N-glycan processing in filamentous fungi, however, its biological significance keeps unclear. [Objective] To evaluate the effects of the N-glycan processing on cell growth and protein secretion in T. reesei, A. fumigatus MsdS was introduced into T. reesei to change the glycoform on mature secreted proteins. [Methods] The recombinant plasmid haboring the msdS gene was constructed and transformed into T. reesei to obtain the msdS-expressing strain Tr-MsdS. The phenotypes, N-glycome, protein secretory pathway and cellulase activity were analysed. [Results] The msdS-expressing strain Tr-MsdS produced a major glycoform of Man₆GlcNAc₂ on its secreted glycoproteins, instead of Man₈GlcNAc₂ in the parent strain. Although the cell wall content of msdS-expressing strain Tr-MsdS was changed, it appeared that the cell wall integrity was not affected. However, phenotypes such as increased conidiation, multiple budding and random branching were observed in strain Tr-MsdS. In addition, expression of MsdS in T. ressei also affected protein secretion and increased the acivities of cellulose and β-mannan degradation by 9.9% and 32.2% at 50℃, respectively. [Conclusion] Our results indicate that the N-glycan processing plays an important role in protein secretion in T. reesei, especially cellulases. Also, our results provide a new strategy to improve cellulases production by interfering the N-glycan processing in T. reesei.

Abstract:[Background] Cellulose is one of the main raw materials for biotransformation to solve energy problems. Furfural, existing in the cellulose hydrolysate, which seriously affects the growth inhibition of strains, must be detoxified before it can be applied in fermentation. Therefore, improving the strain tolerance of furfural is the key to solve the practical production application of cellulose hydrolysate. [Objective] Saccharomyces cerevisiae is a major industrial strain of cellulose hydrolysate fermentation, but it could not tolerant high furfural, and the strain with high furfural tolerance was obtained by molecular modification. [Methods] In this study, the furfural relevant transcription factors obtained from Candida glycerinogenes, CgSTB5, CgSEF1 and CgCAS5, were performed to modify S. cerevisiae to investigate their effects on furfural tolerance and apply to the ethanol fermentation of undetoxified cellulose. [Results] Expression of CgSTB5 or CgSEF1 could increase the furfural tolerance more than 25% in the spot plate experiment. The furfural degradation performance was significantly improved. The growth retardation period was shortened. The production efficiency of undetoxified cellulosic ethanol fermentation with S. cerevisiae W303/p414-CgSTB5 was increased by about 12.5%. [Conclusion] Both CgSTB5 and CgSEF1 could improve furfural tolerance in S. cerevisiae, which contribute to the improvement of the fermentation performance of undetoxified cellulosic ethanol in S. cerevisiae.

Abstract:[Background] Stachybotrys longispora FG216 is a rare marine fungus, and its secondary metabolite FGFC1 has fibrinolytic activity. The genome sequence analysis of S. longispora FG216 will enrich and promote the basic research and applied research of synthetic biology of marine microbial functional genes and secondary metabolites. [Objective] To detected the genome sequence of S. longispora FG216, analyze the biological function and homologous similarity relation of the genome, and reveal the related secondary metabolite genes of the fibrinolytic active compound FGFC1. [Methods] Based on Illumina HiSeq high-throughput sequencing platform, S. longispora FG216 strain was de novo sequenced. SSPACE, Augustus and other software were used to perform assembly, encoding gene prediction, gene function annotation, species collinear analysis and prediction of FGFC1 secondary metabolite synthesis gene cluster. [Results] The total sequence length of S. longispora FG216 genome was 45 622 830 bp, and a total of 605 scaffolds were obtained, with a GC content of 51.31%. Annotations predicted 13 329 coding genes and 169 non-coding RNAs. The genome sequencing data submitted to the National Microbiology Science Data Center numbered NMDC60016264, of which 13 053, 8 422, 8 460, 7 714 and 2 847 genes can be matched with the annotation information in NR, KEGG, KOG, GO and CAZy databases respectively. According to comparative genomics analysis, Stachybotrys is conservative, with core genes accounting for 71.44% of the total gene families. S. longispora FG216 and S. chlorohalonata IBT 40285 had the highest similarity. At the same time, 101 secondary metabolite synthesis gene clusters were predicted, among them 18 gene clusters matched the known compounds. Under the antiSMASH predicts that Cluster 57 is a gene cluster encoding the synthesis of the parent nuclear structure of FGFC1 isoindolinone, and the similarity of the gene cluster with S. chlorohalonata IBT 40285 is 40%. [Conclusion] The genome information of the rare marine fungus S. longispora FG216 has been uploaded to the National Microbial Science Data Center for public use, which provides important reference for the study of Stachybotrys species. Cluster 57 was also found to be the partial encoding gene of the parent nucleus of the fibrinolytic active compound FGFC1, a secondary metabolite of S. longispora FG216.

Abstract:[Background] With the environmental features of high pressure, low temperature and no light, deep sea areas contain abundant and characteristic microbial resources. [Objective] This study aims to isolate and screen aerobic denitrification bacteria strains with high denitrification efficiency from deep sea sediments, and reveal their denitrification characteristics, so as to provide material basis for the development of water denitrification microtechnology. [Methods] In this paper, deep-sea sediments from 10 stations in the East Pacific Ocean, South Atlantic Ocean and Southwest Indian Ocean were used as research materials. Two consecutive rounds of enrichment culture were carried out with inorganic nitrogen at 28℃. The strains capable of removing ammonia nitrogen, nitrite nitrogen and nitrate nitrogen were isolated, purified and screened by their qualitative denitrification capacities. The obtained strains were preliminary identified and classified through morphological and 16S rRNA gene sequence analysis. The growth and denitrification performance of the obtained strains were quantitatively studied in the media with ammonia nitrogen, nitrite nitrogen and nitrate nitrogen as the only nitrogen source, respectively. [Results] A total of 49 aerobic denitrifying bacteria were isolated from 10 deep-sea sediment samples, of which 3 strains, designated Pseudomonas sp. G111, Pseudomonas sp. G112 and Dietzia maris W023a, showed high denitrifying efficiency under aerobic conditions. Strains G111 and G112 shared 16S rRNA gene similarities of 99.2% with Pseudomonas bauzanensis BZ93^T. The 16S rRNA gene similarity between strain W023a and Dietzia maris ATCC 35013^T was 99.9%. After strains G111, G112 and W023a were cultured for 48 hours, their removal rates of ammonia nitrogen were 98.0%, 85.2% and 97.6%, respectively; their removal rates of nitrite nitrogen were 71.9%, 67.5% and 34.7%, respectively; their removal rates of nitrate nitrogen were 66.0%, 52.6% and 34.7%, respectively. Strains G111, G112 and W023a are three heterotrophic nitrifying-aerobic denitrifying bacteria. These strains could reduce nitrite or nitrate to nitrogenous gases by using aerobic denitrification, and convert ammonia nitrogen to nitrogenous gases by using heterotrophic nitrification-aerobic denitrification. [Conclusion] In this study, three strains of highly efficient aerobic denitrifying bacteria were isolated from deep-sea sediments, and the obtained strains have application potential in water purification, sewage treatment, ecosystem restoration.

Abstract:[Background] Hadal trench sediments have been reported to harbor abundant microbial cells and have active microbial carbon turnover. Therefore, isolation of microbial resources from trench sediments is of great significance for understanding the biogeochemical cycle and energy metabolism in the hadal trench. It is well known that aromatic compounds are widely spread in the environments. Omics analysis has revealed possible presence of aromatics-degrading microbes in the trench environment. However, there were little reports on the pure culture of trench derived aromatics-degrading microbes nor the possible degradation mechanism.[Objective] To isolate and cultivate bacteria capable of aromatics degradation from the Mariana Trench sediments and characterize its degradation pathway.[Methods] 4-Hydroxybenzoate (4HBA) was used as the sole carbon source to isolate bacteria from the Mariana Trench sediment. Morphological observation and phylogenetic analysis were used to identify the isolate. Growth curves were conducted to verify its optimum growth conditions and degradation ability. The metabolic intermediates during whole cells transformation and the activity of crude cell extracts against the substrate were analyzed, based on which a putative catabolic pathway was reconstructed. [Results] An aerobic bacterium was isolated from the trench sediment and belonged to Citricoccus genus, according to the phylogenetic analysis of its 16S rRNA gene. Colonies of this bacterium, designated Citricoccus sp. strain NyZ702, on LB agar plate were lemon yellowish, opaque, swell, neat edge and round with a diameter of 1—2 mm after 4-day cultivation at 30℃. Cells observed with Scanning Electric Microscope were spherical with a diameter of 0.4—0.6 μm without flagella. The strain was halotolerant with an optimal salt concentration of 2%—8% (W/V). The strain was able to utilize 4HBA as the sole carbon source and protocatechuate was detected as its catabolic intermediate, indicating it degraded 4HBA through the protocatechuate pathway. The cell extracts of this strain showed monooxygenase activity against 4HBA using NADPH as the cofactor. [Conclusion] A 4HBA degrading bacterium, Citricoccus sp. strain NyZ702 was isolated from the trench sediment and its 4HBA degradation was via protocatechuate as the intermediate metabolite. This study would enrich the microbial resources derived from the hadal trench, providing a certain theoretical foundation for the future research of aromatics degradation in the hadal trenches.

Abstract:[Background] Reducing Cr(VI) to Cr(III) by dissimilatory Fe(III)-reducing bacteria is an important way for remediation of chromium pollution. [Objective] This work examined the characteristics of Cr(VI) reduction by biogenic magnetite particles using dissimilatory Fe(III)-reducing bacterium Clostridium butyricum LQ25. [Methods] Strain LQ25 was grown under Fe(III)-reducing conditions and was used to produce biogenic magnetite particles using glucose as electron donors and insoluble iron hydroxide as the electron acceptor. The efficiencies of Cr(VI) reduction under Fe(III)-reducing conditions were examined with different Cr(VI) concentrations (5, 10, 15, 25 and 30 mg/L) added to the medium respectively. [Results] In the presence of iron hydroxide, strain LQ25 could grow within the set Cr(VI) concentration range with Cr(VI) reduction efficiency of 63.45%±5.13% at Cr(VI) concentration of 15 mg/L. When adding biogenic magnetite particles, Cr(VI) reduction efficiency of 87.73%±9.12% was obtained, increased by 38% compared with the control. Cr(VI) reduction by biogenic magnetite particles was decreased obviously with increasing pH solutions from 2.0 to 8.0. At pH 2.0, the reduction rate of Cr(VI) by biogenic magnetite particles was the highest, almost 100%. There were many pores on the surface of biogenic magnetite particles under scanning electron microscope. Biogenic magnetite particles showed XRD peaks include Fe(II) existed in the form of Fe(OH)₂.[Conclusion] These results indicated that dissimilatory Fe(III)-reducing bacteria and the biogenic magnetite particles could reduce Cr(VI), which will provide the evidence for the application of dissimilatory Fe(III)-reducing bacteria into the reduction of Cr(VI).

Abstract:[Background] Xylan which widely exists in lignocellulosic biomass is the most abundant hemicellulose in the world. It is an effective way for the resource and energy utilization of lignocellulosic biomass to utilize enzyme producing microorganisms to ferment lignocellulosic biomass. [Objective] The aim of this experiment was to obtain materials for producing multiple feed additives from multi fiber agricultural and forestry wastes by screening and identifying xylanase producing strains, analyzing enzymatic characteristics and optimizing fermentation conditions. [Methods] The xylanase producing strains were screened from the soil of Qinghai-Tibet Plateau. The strain XC70 were identified by morphological observation and rDNA ITS sequence analysis. The enzymatic characteristics, growth and enzyme production attributes of the strain were analyzed, and the fermentation conditions were optimized by single factor method and orthogonal test method. [Results] The strain XC70 was identified as Penicillium oxalicum by morphological and molecular biological methods. The results showed that the optimal reaction conditions of xylanase produced by XC70 were pH 5.0, 70℃, the xylanase had good stability when the temperature was lower than 50℃, and it showed some acid resistance. Na⁺ and K⁺ promoted the xylanase activity (P<0.05). After 54 h of fermentation, the biomass and enzyme activity of supernatant reached the peak. Through single factor method and orthogonal test method, the optimal fermentation conditions were determined as follows:peptone 7 g/L, corn straw 50 g/L, KCl 4 g/L, initial pH 4.0, 28℃, shaking speed 200 r/min, inoculum size 2%. Under these conditions, the xylanase activity reached 1 489.33 U/mL, which was three times higher than that before optimization. [Conclusion] The strain XC70 screened from the soil of Qinghai-Tibet Plateau has certain acidic xylanase-producing capacity, which can be used to degrade multi fiber materials and develop new feed additives, and this has certain application potential and development value.

Abstract:[Background] Stellera chamaejasme L. is one of the landmark plants of natural grassland degradation in China. The root endophytic microorganisms play an important role in the process of invasion. It is of great significance to explore the root endophytic microbial community to the utilization beneficial microbial resources, and to prevention and control of the Stellera chamaejasme L. [Objective] The purpose of this study was to explore the endophytic fungi and bacteria composition and diversity in the roots of Stellera chamaejasme L., a poisonous weed in different alpine grasslands at the eastern foot of Qilian Mountains in Gansu province. [Methods] The composition and diversity of endophytic fungi and bacteria in the roots of Stellera chamaejasme L. in different alpine grasslands at the eastern foot of Qilian Mountains in Gansu province were analyzed by Illumina MiSeq high-throughput sequencing technique. [Results] The results showed that Ascomycota in different alpine steppe was dominant in fungi in the roots of Stellera chamaejasme L. from different alpine grasslands. Cyanobacteria, Proteobacteria and Actinobacteria belong to the dominant groups in root endophytic bacteria. The operational taxonomic unit (OTU) number and Shannon diversity index of fungi and bacteria in G4 sampling site were higher than those in the other four sampling sites. Principal coordinate analysis (PCoA) showed that there were some differences among endophytic fungi and bacteria communities in the roots of Stellera chamaejasme L. in different alpine grasslands. One-Way ANOVA analysis and Pearson correlation analysis showed that the community structure of Stellera chamaejasme L. fungi had little correlation at the genus level, while the bacteria had a certain correlation at the genus level.[Conclusion] The purpose of this study is to further develop and utilize the endophytic fungi and bacterial resources and provide theoretical reference for elucidating the community structure and ecological function of endophytic flora of Stellera chamaejasme L.

Abstract:[Background] In recent years, the application of quorum quenching (QQ) technology in the prevention and control of membrane biofouling has received extensive attention. However, limited QQ bacteria have been successfully isolated and identified. More efficient signal molecule degrading bacteria awaits identification and further investigation. [Objective] Isolate more efficient QQ bacteria from native real membrane bioreactor (MBR) activated sludge and extending QQ bacteria resources. [Methods] Agrobacterium tumefaciens A136 was used as the reporter strain, to test isolated strain's C8-HSL (N-octanoyl-DL-homoserine lactone) degradation. Reporter Chromobacterium violaceum VIR24 was used to quantify signal molecules degradation of QQ bacteria. 16S rRNA gene sequencing was used to identify isolated bacteria, phylogenetic trees taxonomic of the isolates were then constructed. Scanning electron microscopy (SEM) was used to determine the morphology of bacteria. Coculture of QQ strain and typical biofilm forming bacteria was conducted to analyze biofilm inhibition ability of isolates. QQ beads was prepared using polyvinyl alcohol and sodium alginate. [Results] Six QQ strains were successfully isolated and identified, among which a Gram negative rod strain Delftia sp. JL5 showed the highest efficiency in C8-HSL degradation. Besides, our results showed that JL5 significantly inhibited biofilm formation of both Pseudomonas aeruginosa PAO1 and Pantoea ananatis SK-1, which are two typical N-acyl-homoserine lactones (AHL) dependent biofilm forming bacteria. Furthermore, JL5 remained high AHL degrading activity after being entrapped in QQ beads. Its AHL degrading efficiency was higher than the widely reported Rhodococcus sp. BH4. [Conclusion] We successfully isolated QQ bacteria. The isolates showed high C6/C8-HSL degrading ability. The bacteria inhibited biofilm formation efficiently. This research set solid foundation for further application of QQ bacteria in biofouling control.

Abstract:[Background] Some bacterial DNA skeleton undergo phosphorothioate modification, sulfur binding domain (SBD) can specifically recognize this physiological modification. Unlike most SBD-HNH di-domain nucleases, SET and RING-associated (SRA) domain, specifically recognize DNA 5-methylcytosine (5mC), is inserted between SBD and HNH domains of ScoMcrA. The crystal structures show that single SBD is a monomer and SBD-SRA is a dimer.[Objective] The effects of the presence of SRA domain on the binding of sulfur modified DNA by SBD, and the way SRA domain affect the phosphorothioated DNA recognition. [Methods] Electrophoresis mobility shift assay (EMSA) was applied to compare binding affinity of SBD and SBD-SRA to sulfur modified DNA respectively. The key amino acid residues involved in dimerization of SBD-SRA were mutated to examine the binding affinity of mutant proteins to phosphorothioated DNA. [Results] Compared with SBD domain alone, the di-domain protein SBD-SRA showed enhanced affinity to sulfur modified DNA. The single point mutation of ten amino acid residues at the dimer-forming interface of SBD and SRA domain seldom affect its binding affinity to sulfur modified DNA. By comparison, L₂₆₁LGET₂₆₅ are simultaneously mutated to A₂₆₁AAAA₂₆₅ on SBD-SRA, the binding affinity of the mutant to sulfur modified DNA decreased to a level similar to that of SBD. [Conclusion] According to EMSA results, we primarily came to the conclusion that SRA domain can improve the binding ability of SBD to sulfur modified DNA in SBD-SRA di-domain protein; L₂₆₁LGET₂₆₅ is the key amino acid sites in the SRA domain that affects the binding ability of SBD to sulfur modified DNA.

Abstract:[Background] The toxin-antitoxin system is widespread in microorganisms and plays an important role in their defense against adverse environments. [Objective] we used Yersinia pseudotuberculosis (Yptb) as the meterial to investigate the mechanism and biological functions of the Phd-Doc toxin-antitoxin system. [Methods] The Phd-Doc toxin-antitoxin system encoded in Yptb was identified by bioinformatics, and subsequently confirmed by toxicity experiment, gene expression analysis and protein interaction assay. To study the biological function of Phd-Doc toxin-antitoxin system in Yptb, the differences between the wild type strains and mutant strains were compared through antibiotic stressing, oxygen stressing and biofilm formation experiments. [Results] Bioinformatics analysis showed that a pair of Phd-Doc toxin-antitoxin system is present in Yptb, and subsequent experiments confirmed that these proteins are co-transcripted and display high affinity. The cell morphology was changed and cell growth was inhibited when the Doc toxin protein expressed in Escherichia coli, and these changes were rescued by the antitoxin protein Phd. The transcription level of phd-doc was significantly up-regulated under stress conditions, especially under the chloramphenicol stress. Deletion of phd-doc significantly affected the biofilm forming ability of Yptb but did not influence the cell growth. [Conclusion] The functional identification of the Phd-Doc toxin antitoxin system in Yptb is of great significance for a better understanding of the colonization and response mechanism of microorganisms under the changing external environment.

Abstract:[Background] Starmerella bombicola, as an unconventional yeast strain, has attracted wide attention owing to its ability to produce sophorolipids biosurfactant. However, its expression system is not well defined, which limits the development of metabolic engineering. [Objective] A panel of endogenous promoters were cloned and characterized from S. bombicola. [Methods] In this study, through the comparative analysis of the whole genome of S. bombicola and 9 target genes, combined with the promoter prediction website, a series of promoter candidate sequences were screened and obtained, and SbGFP (codon-optimized yeast enhanced green fluorescent protein for S. bombicola), as the reporter gene, was integrated and expressed in S. bombicola. The promoter strength was identified by analyzing the intensity of green fluorescent protein and its transcriptional levels. [Results] When glucose and colleseed oil were respectively used as the sole carbon source, the promoters P_TEF1 and P_GPD showed higher transcription levels under both conditions. The promoters P_CYP52M1, P_UGTA1, P_UGTB1, and P_MOB had weak transcriptional activity when colleseed oil was used as the sole carbon source, but no transcriptional activity was detected when cultured with glucose. It was speculated that they were colleseed oil-inducible promoters. Transcriptional level of SbGFP was further analyzed by real-time fluorescence quantitative PCR (RT-qPCR), the result was consistent with the expression level of SbGFP.[Conclusion] A panel of different promoters were screened and characterized, which would further enrich its expression elements and lay a theoretical foundation for the metabolic engineering for S. bombicola.

Abstract:[Background] As one of the unicellular green algae, Chlorella sp. can accumulate a variety of value-added metabolites under different culture conditions. These metabolites could be used as a good feedstock to produce cosmetics, foods, health products, medicines, etc. However, their large-scale industrial production is restricted because of low productivity of metabolites in the algal cells and the difficulty of separating them by economically viable methods. [Objectives] To study the effects of sodium acetate on biomass production of Chlorella sorokiniana, and to analyze its regulatory role in the production of algal metabolites. [Methods] Different concentrations of sodium acetate (1.0, 2.0, 3.0, 4.0 and 5.0 g/L) were added to the cultures of C. sorokiniana. Growth and metabolites of this alga were determined during algae cultivation to study the regulatory role of sodium acetate. [Results] Biomass concentration of C. sorokiniana was 5.2 times higher than that in the controls, when this alga grew in the cultures with 3.0 g/L sodium acetate. Although no obvious changes in contents of proteins were observed, contents of lipids and carotenoids were 2.4 and 1.2 times greater than that in the controls, respectively, when the alga grew in the cultures with 3.0 g/L sodium acetate. In addition, contents of polysaccharides and chlorophyll a decreased, which were only 54.6% and 54.4% of that in the controls, respectively. [Conclusion] Sodium acetate not only affected the growth of C. sorokiniana, but also regulated its metabolic process. These results would provide theoretical basis and technical data for further exploring the regulatory role of sodium acetate in growth and metabolic process of C. sorokiniana.

Abstract:[Background] Gardenia jasminoides Ellis is a perennial evergreen shrub. After years of continuous planting, it will lead to deterioration of the soil micro-ecological environment, aggravation of pests and diseases, and reduced quality. It was found that intercropping was an effective cultivation method in production to improve soil microflora, nutrient and enzyme activity. [Objective] In this study, the dynamic changes of the rhizosphere soil microflora, enzyme activity and nutrients of gardenia under different intercropping patterns were studied, in order to provide a theoretical basis for revealing the soil microecology mechanism of cultivation measures to improve the soil ecological environment and enhance the yield of gardenia.[Methods] To understand the effects of different intercropping patterns on the rhizosphere microecology, three-year-old gardenias were selected for field experiment. Random block design was adopted. Gardenia single cropping and Gardenia/Bletilla striata, Gardenia/Lysimachia christinae, Gardenia/Belamcanda chinensis three intercropping treatment were set, using Gardenia rhizosphere soil as the research material, sampling throughout the growth periods. Illumina high-throughput sequencing technology was used to determine the sequences of bacterial 16S rRNA gene V3—V4 and fungal rDNA ITS1—ITS2, and the physical and chemical properties of the soil in each period were determined, so as to clarify the intercropping of different crops on the gardenia rhizosphere microbial community and soil physical and chemical properties with the growth period.[Results] In the whole growth process of gardenia, the relative abundance of Proteobacteria and Acidobacteria in the rhizosphere bacterial community were 39% and 18%, respectively, which were the dominant bacteria. In the fungal community, the relative abundance of Ascomycota, Basidiomycota and Mortierellomycota accounted for 51%, 22% and 19%, respectively, which were the main fungal groups. In the fruit expansion stage, compared with single cropping, Gardenia/Belamcanda and Gardenia/Bletilla striata can significantly increase the Shannon index of soil bacterial communities by 6.55% and 3.45% (P<0.05), respectively, but there was no significant difference at other stages. In terms of rhizosphere fungi, Gardenia/Belamcanda does not significantly reduce the diversity during the full blooming period, while intercropping with Lysimachia or Bletilla will significantly decrease; In the fruit expansion stage, Gardenia/Belamcanda and Gardenia/Bletilla intercropping can significantly increase the Shannon index of the rhizosphere soil fungal community by 29.19% and 9.12%, respectively. In terms of soil nutrients, The contents of organic matter, total N and available P in rhizosphere soil of gardenia monoculture were higher, while the contents of alkaloid N and available K were lower than the three intercropping treatments. In terms of soil enzymes, except for the acid protease, the activities of the other soil enzymes of the single-cropped gardenia are at a lower level. Correlation analysis between soil physical and chemical properties and rhizosphere microbial diversity showed that the bacterial diversity index Shannon was significantly positive correlated with rhizosphere soil organic matter, available P, and extremely significantly positive correlated with pH (P<0.01); The fungal diversity index Shannon was significantly negative correlated with total K, urease and catalase in rhizosphere soil, and significantly positive correlated with available K, sucrase, acid phosphatase, and acid protease activities. The comprehensive scores of soil fertility in the blooming stage and fruit expansion stage showed that the soil fertility scores of Gardenia/Belamcanda was the highest, and that of Gardenia/Lysimachia was the lowest.[Conclusion] Reasonable intercropping can improve the microbial community structure in the rhizosphere, and improve the comprehensive soil fertility. The soil fertility comprehensive score of Gardenia/Belamcanda is the highest, they may be the desirable intercropping patterns for Gardenia cultivation.

Abstract:[Background] In the process of subculture, Flammulina filiformis strains will degenerate, which affects the yield and quality of F. filiformis strains. [Objective] The physiological and biochemical characteristics of the mycelia of the degenerative strains of F. filiformis were studied in order to select the degenerate strains of F. filiformis. [Methods] This experiment by F. filiformis original strain (H) and degenerated strains (T) as the research object, measuring the different carbon source culture medium on physiological and biochemical characteristics of mycelia and superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity, and determination of mycelia in the cultivation of laccase (Lac) and the activity of the manganese peroxidase (MnP), recorded the mycelia recovery after scratching the fungi.[Results] The mycelia growth rate of T in each carbon source was lower than that of H, the levels of oidiospore were between 3—4, the activities of SOD and CAT activity were lower than that of H, and the activity of MnP and Lac were the same as H at the 5th day, and lower than H at the 10th, 15th and 20th days. The recovery time of T was longer than H, and the growth thvigor of the restored mycelia was less dense than H. [Conclusion] By exploring the physiological and biochemical characteristics of the mycelium of the original strain and the degenerated strain of F. filiformis, it provides a theoretical basis for judging whether the strain of F. filiformis is the degenerated strain.

Abstract:[Background] Hydrogen sulfide (H₂S) is a toxic and harmful gas released in the process of livestock production, which seriously harmed the health of livestock and human beings. Therefore, the degradation of hydrogen sulfide, especially the transformation of hydrogen sulfide by biological oxidation, has become a current research focus. [Objective] In order to screen the highly efficient sulfur-oxidizing strains and study its biotransformation. [Methods] Using fresh feces collected from a chicken farm in Changchun city as the material, sulfur-oxidizing strains were isolated and identified. The growth conditions were optimized by using single-factor analysis, the biotransformation efficiency was studied, and the mRNA expression levels of soxY and soxZ genes were detected. [Results] A highly efficient sulfur-oxidizing strain, JF9, was successfully obtained, which was preliminarily identified as Alcaligenes faecalis. Optimal growth conditions:substrate concentration 0.5 g/L, temperature 35℃, initial pH 7.0. Under these conditions, the removal percentage of Na₂S was over 94%. The strain JF9 had soxY and soxZ genes, and their transcription levels were greatly induced by sulfur substrates (P<0.05). [Conclusion] The isolated strain Alcaligenes faecalis had a considerable ability to degrade sulfide, and the sulfur-oxidizing genes were highly expressed during the desulfurization process.

Abstract:[Background] Walnut blight is a serious bacterial plant disease caused by two pathogens, it lacks effective biological control method at present. [Objective] To isolate and screen antagonistic actinomycetes strains with inhibitory activity on walnut blight from the rhizosphere soil of walnut tree. [Methods] Actinomycete was isolated by using dilution plate coating. The methods of agar disk and improved Oxford-cup test were applied to screen antagonistic strains against Xanthomonas campestris pv. campestris and Pantoea agglomerans. The morphological, physiological, biochemical characteristics combined with 16S rRNA gene sequences were used for classification and identification. The antibacterial spectrum and the biocontrol effect of the cell-free fermentation broth were evaluated. [Results] Strain WMF106 had antagonistic effects on X. campestris pv. campestris with inhibition zone diameter of 2.38 cm and on P. agglomerans with inhibition zone diameter of 1.82 cm. The inhibition zone diameter of cell-free fermentation broth on X. campestris pv. campestris and P. agglomerans were 1.75 cm and 1.55 cm, respectively. Based on the morphological and physiological characteristics and 16S rRNA gene sequence analysis, strain WMF106 was identified as Streptomyces caeruleatus. The antibacterial spectrum experiment results indicated that Streptomyces caeruleatus WMF106 also showed biological activities against five species of plant pathogenic fungi (Fusarium oxysporum, Fusarium solani, Colletotrichum capsici, Botrytis cinerea and Colletotrichum gloeosporioides), and five species of indicator bacteria (Aspergillus niger, Monilia albican, Escherichia coli, Staphlococcus aureus and Pseudomonas aeruginosa). In addition, the biocontrol experiment in vitro showed that the cell-free fermentation broth of strain WMF106 had worthy prevention of walnut blight caused by X. campestris pv. campestris and P. agglomerans, and the biocontrol effects were 77.44% and 58.33%. [Conclusion] Strain WMF106 could be used for controlling the walnut blight, and have a wonderful potential for application.

Abstract:[Background] The mining of bacterial resources with the capabilities of both nicotine degradation and plant growth-promoting would contribute to soil quality protection and green cultivation. [Objective] To analyze the bacterial diversity in the rhizosphere of flue-cured tobacco and screen the plant growth-promoting bacteria that can degrade high concentration of nicotine. [Methods] Pure culture is applied to isolate nicotine-degradation strains with selective medium. The diversity and phylogeny of rhizobacteria were analyzed by transcription factor amplification (BOXA1R-PCR), 16S rRNA gene sequencing and phylogeny analysis. The PGPR characteristics of the nicotine-degradation strains were screened via determination of indole-3-acetic acid (IAA) production, phosphorus solubility, antimicrobial activity and other indexes. The growth-promoting effect of the isolates were further confirmed by pot experiment. [Results] Totally, 58 strains of nicotine-degradation bacteria were isolated, and 11 typical strains with different BOXA1R-PCR fingerprints were selected for 16S rRNA gene sequencing. Based on the phylogeny analysis, the 58 strains were belonged to four genera including Bacillus, Pseudomonas, Raoultella and Brevundimonas, with Bacillus as the predominant genus. Of the 58 isolates, 48.28% produced IAA, 27.59% were able to dissolve phosphorus, 37.93% could degrade cellulose. Based on the PGPR abilities, three isolates including G2-13, G2-3 and HT2-8 were used to perform pot experiment to further confirm their potential growth-promoting ability. The results showed that when compared to the control, G2-13 could increased the plant height and aboveground fresh weight by 33.05% and 53.32%, respectively. [Conclusion] The tobacco rhizosphere harbors plenty of nicotine-degradation bacterial strains with PGPR capability and have potential application prospects in plant cultivation strains.

Abstract:[Background] Chemotaxis of microorganisms mediated by root exudates is an important prerequisite for root colonization and function of plant growth-promoting rhizobacteria (PGPR), and further understanding of this process is of great significance to understand the colonization mechanism of the strains. [Objective] To study the signal substances in root exudates of potato which can promote the colonization of PGPR QHZ3 in rhizosphere. [Methods] The some phenolic acids in potato root exudates were identified by high performance liquid chromatography (HPLC). the chemotaxis of potato root exudates and different phenolic acids to isolate QHZ3 were compared using the semi-solid plate method and capillary-like method. the effects of different phenolic acids on biofilm formation of QHZ3 were observed by crystal violet staining. [Results] Phenolic acids in potato root exudates mainly included fumaric acid, p-hydroxybenzoic acid, ferulic acid and cinnamic acid. The semi-solid plate method shows that both the root exudates of potato and the four phenolic acids mentioned above had chemotactic effects on strain QHZ3, and the chemotactic effect of fumaric acid was the strongest. capillary-like quantitative test showed that the chemotactic effect of phenolic acids on strain QHZ3 was different at different concentrations. The chemotactic effect of middle and high concentrations of fumaric acid (25—100 μmol/L) and low concentration of ferulic acid (10 μmol/L) on strain QHZ3 was the strongest. Crystal violet staining showed that 120—240 μg/ml potato root exudates, 50—75 μmol/L fumaric acid and 100 μmol/L p-hydroxybenzoic acid could significantly promote biofilm formation of QHZ3, while ferulic acid and cinnamic acid had no significant effect on biofilm formation. [Conclusion] Both root exudates and phenolic acids could mediate the chemotaxis of strain QHZ3 to form biofilm in potato rhizosphere, but the effects of four phenolic acids were different. The chemotaxis of fumaric acid and ferulic acid was significant, while fumaric acid and p-hydroxybenzoic acid had significant effects on the formation of biofilm.

Abstract:[Background] Cyanobacteria are key contributors to biological nitrogen fixation in aquatic and terrestrial ecosystems. [Objective] To increase our knowledge of nitrogen-fixing cyanobacteria in paddy soil and obtain culturable nitrogen-fixing cyanobacterial strains for further studies. [Methods] Three paddy soils with different nitrogen fixation capabilities were selected to isolate nitrogen-fixing cyanobacterial strains with BG11-N medium. The morphological characteristics of newly isolated strains were observed. The nifH gene amplification of genomic DNA was used to clarify their nitrogen fixation potential, acetylene reduction assay and ¹⁵N₂ tracer method were used to quantitatively determine their nitrogen fixation activity. Preliminary identification of the newly isolated strains were determined through 16S rRNA gene sequence comparison. [Results] Seven autotrophic strains were isolated and purified with BG11-N medium under light conditions. All cells were round or elliptical, uniseriate, unbranched, filamentous and moniliform, and formed mat-like colonies on solid medium. The newly isolated strains grew well in BG11-N medium, were positive for nifH gene amplification of genomic DNA, had high nitrogen fixation activity measured by acetylene reduction assay and ¹⁵N₂ tracer method, and had capabilities of siderophore production. Through 16S rRNA gene sequence comparison and morphological features, all the seven strains could be preliminarily identified as belonging to Nostocaceae. [Conclusion] The cyanobacterial (Nostocaceae) strains that plays important role in biological nitrogen fixation were isolated from paddy soil. The culturable nitrogen-fixing cyanobacterial strains have high nitrogen fixation and siderophore production ability, which could be used as microbial resources for further studies and have potential research and application value.

Abstract:[Background] Bacterial fruit blotch of cucurbits is a serious seed-borne bacterial disease, and its pathogen is Acidovorax citrulli. So far, the understanding of the interaction mechanism between pathogen and its hosts is extremely limited. Cucumber, a model plant in the Cucurbitaceae family, is one of the hosts and easy to be infected. The transcriptome analysis of the interaction of Ac-cucumber can lay an important foundation for exploring the interaction mechanism of Ac and its host.[Objective] To analyze the responses of both Ac and cucumber under the interaction. [Methods] 6 d cotyledons of cucumber were inoculated with bacteria cell suspension, and 48 h cotyledons post inoculation were sampled for transcriptome sequencing. The RNASeq technology was used to analyze the gene expression features of Ac FC440 strain interacting with 9930 cwltivar of cucumber. [Results] The sequencing data shows that the correlation between different replicates of each sample was strong, and the comparison rate with the reference genome was over 95%. Cluster analysis indicates that the expression patterns of the control were opposite to that of the treatment, and the sample processing achieved a certain effect, indicating that the overall quality of the data was high. Six differentially expressed genes were selected for RT-qPCR verification, and the results showed that the expression level of the six genes were basically consistent with the transcriptome results, indicating that the transcriptome sequencing results were relatively reliable. Under 48 h interaction between Ac and cucumber, there were 1 618 genes and 8 698 genes expressed differentially, respectively. Gene ontology (GO) analysis shows that differential genes of bacteria are significantly enriched in the cell membrane (37.5%) and membrane part (27.0%) in cellular components, oxidation-reduction process (66.7%) in biological process and hydrolase activity (66.5%) in molecular function. Differential genes of cucumber are significantly enriched in plastid (22.2%) and chloroplast (21.3%) in cellular components, catalytic activity (70.0%) in molecular functions and carbohydrate derivative metabolic process (32.2%) in biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that pathogenic related genes in bacteria were significantly enriched in the quorum sensing and bacterial chemotaxis pathways, and the genes of quorum sensing were down-regulated more than others. Genes regulating expression of calcium-dependent protein kinase (CDPK), calmodulin and calmodulin-like (CaMCML) and respiratory burst oxidase homologne (Rboh) in cucumber were up-regulated, and genes regulating expression of phenylalanine ammonia-lyase (PAL) and glutathione S-transferase (GST) are the most abundant ones and up-regulated in the corresponding metabolic pathways. [Conclusion] The quality of transcriptome sequencing data of Ac and cucumber interaction was high. Quorum sensing correlated to the pathogenicity of FC440 strain in Ac. The main path that cucumber's defense reaction against to Ac infection is activated by Ca²⁺ signal. PAL and GST play an important role in resistance of cucumber to Ac. This study lays the foundation for further exploration on the interaction mechanism between Ac and its host.

Abstract:[Background] Natural production derived from microorganisms are one of the hot topics in the research and development of new pesticides. [Objective] To isolate and screen soil fungi producing potential herbicidal metabolites. [Methods] Petri dish filter paper culture method was adopted to determine the inhibition effect and stability of fermentation broth on the growth of plant seedlings, and the active strain was identified by microscopic observation and rDNA ITS sequence analysis. [Results] Of 30 fungal strains isolated from soil, the fermentation broth of isolated strain L-27 had significant inhibitory effect on wheat seedling, with inhibition rates 79.4% and 67.3% on root and stem length. Based on colony morphology, micromorphology and rDNA ITS gene sequences analysis, the isolated strain L-27 was identified as Aspergillus tubingensis. Further, strain L-27 fermentation broth completely inhibited seedling growth of Amaranthus retroflexus, Portulaca oleracea, Echinochloa crusgalli and Ipomoea purpurea except for 77.8% reduction of stem length on I. purpurea. The inhibitory effect of strain L-27 fermentation broth on wheat seedling growth had high stability to temperature, pH and UV light radiation, with inhibition rate 100% and 73.8% on root and stem length even heated at 120℃ for 20 min, 100% and 100% at pH 2.0—12.0 for 1 h, and 100% and 84.3%—91.7% under UV light radiation for 5 min to 240 min. [Conclusion] The fermentation broth characteristics of the isolated A. tubingensis L-27 indicated its great potential to develop herbicide.

Abstract:[Background] Fire blight is a bacterial disease caused by Erwinia amylovora, which is harmful to more than 220 plants belonging to more than 40 genera, such as apple, pear, hawthorn and so on. In recent years, fire blight has occurred in many countries, including central Asian countries. E. amylovora has become an extremely high risk microorganism for China, especially for Xinjiang. Fire blight has been listed in the list of first class crop diseases and pests by the Ministry of Agriculture and Rural Areas of China. [Objective] To screen the antagonistic strains of Erwinia amylovora from Xinjiang and improve the ability of Korla fragrant pear to resist the risk of fire blight. [Methods] The antagonistic strains were isolated from the soil of pear orchard and the bacteria stored in laboratory by confrontation plate method. The strains were preliminarily identified by physiological characteristics analysis as well as 16S rRNA gene sequencing, and the antibacterial effect was rescreened by Oxford cup diffusion method. The control effects were tested on two-year-old potted Pyrus betulaefolia seedlings in greenhouse. [Results] Eleven antagonistic strains were obtained, all of which were Gram positive. Nine of them were Bacillus and two were Lactobacillus. The results of confrontation plate experiment showed that Bacillus velezensis JE7, Lactobacillus plantarum LP1 and Lactobacillus plantarum LP2 had strong antagonistic effect, followed by Bacillus velezensis JE4. The control effects showed that Bacillus velezensis JE4 had the best prevention effect in greenhouse, which reached 73%. The physiological characteristics was showed that JE4 and JE7 could tolerate 1%—7% NaCl and could grow at pH 4.0—9.0; LP1 could tolerate 1%—9% NaCl and could grow better at pH 5.0—9.0. [Conclusion] a batch of bacteria against Erwinia amylovora were screened and obtained. Some of the antagonistic strains had significant control effect in greenhouse and strong saline-alkali tolerance.

Abstract:[Background] In northern China, the average temperature in autumn and winter is low, which makes straw more difficult to degrade naturally.[Objective] Screening high efficiency lignin-degrading bacteria of low-temperature, explore enzymatic properties and improve enzyme-producing performance and straw degradation efficiency.[Methods] High efficiency lignin degradation strains were screened by aniline blue method and enzyme activity determination, and the enzymatic production conditions were optimized by single-factor screening and response surface experiment, and the degradation efficiency of straw was determined through solid-state fermentation experiments.[Results] An efficient lignin-degradation strain LS-1 was screened and identified as Stenotrophomonas maltophilia. When the carbon and nitrogen sources of LS-1 were lignin and pepone, pH 8.0, temperature 15℃, time 3 days, the activities of Lip, Lac and Mnp were 23.34 U/mL, 9.37 U/mL and 50.89 U/mL, respectively. The optimal temperature of Lip and Lac was 30℃ and the thermal stability was well, while the optimal temperature of Mnp was 50℃ and the thermal stability was poor, the optimum action environment of the three enzymes was acidic. 0.75 mmol/L Mg²⁺ and 0.5% Tween-20 can promote Lip activity, 1 mmol/L Cu²⁺ and syringic acid can promote Lac activity, and 0.1%—0.5% Tween-20 can promote Mnp activity. After low-temperature solid-state fermentation, the weight loss rate of straw was 18.85%, and the lignin degradation rate was 36.14%, which was about 6 times higher than that of the control group.[Conclusion] This experimental provides high-quality bacterial resources for the efficient degradation of low-temperature lignin and has a well application prospect in straw degradation.

Abstract:[Background] Bacillus is commonly used in microecological preparations next to lactic acid bacteria. However, some Bacillus spp. are not strictly regulated and pose potential safety hazards in application. [Objective] To study the prevalence of Bacillus cereus in Bacillus microecological preparations on sale from all over the country, and reveal the potential risks of the application of B. cereus. [Methods] B. cereus group strains were isolated and identified from the pretreated microecological preparations. Toxicity and antimicrobial resistance of B. cereus group strains were analyzed using whole-genome sequence. The cytotoxicity was evaluated via Cell Counting Kit-8 assay. Antimicrobial susceptibility was determined by broth microdilution methods. [Results] Twenty-three B. cereus group strains were isolated from 50 microecological preparations, which are resistant to ampicillin, lincomycin and tiamulin, and the detection rates of major virulence genes nhe, hbl, cytK, ces were 100%, 30%, 39% and 4%, respectively. All isolates were hemolytic and showed varying degrees of toxicity to Vero cell, 9 of which produced heat-stable toxins. [Conclusion] The widespread of toxin and resistance genes, and apparent observations of hemolytic activity, cytotoxic, and heat-stable toxin production in B. cereus group isolates from Bacillus microecological preparations, pose great threat to food safety and human safety. The findings highlights need for enhanced efforts required in the surveillance of risk factors, virulence factors and antimicrobial resistance in B. cereus from microecological preparations.

Abstract:[Background] Clostridium difficile is an important zoonotic enteric pathogen that can cause antibiotic-associated diarrhea or pseudomembranous colitis in human and various animals. As the main pig producing area in China, Sichuan has not yet reported on the epidemiological investigation of Clostridium difficile from pigs, which brings challenges to the prevention and control of Clostridium difficile from pigs and the guarantee of pork safety. [Objective] To investigate the infection and prevalence of Clostridium difficile from pigs in Sichuan province, and to study the molecular typing of the isolated Clostridium difficile. [Methods] Stool samples (n=110) of pigs suspected of being infected with Clostridium difficile from six farms in Sichuan were collected and isolated on the identification medium of Clostridium difficile by anaerobic culture. Four toxin genes (tcdA, tcdB, cdtA, cdtB) and seven housekeeping genes (adk, atpA, dxr, glyA, recA, sodA, tpi) of Clostridium difficile were amplified by PCR method. Toxin genotyping and multilocus sequence typing were performed on the isolates. [Results] In 110 samples, 20 strains of Clostridium difficile were isolated by Gram staining microscopy and PCR identification, and the isolation rate was as high as 18.18%. Toxin genotyping results showed that three toxin genotypes were obtained, including tcdA⁺tcdB⁺cdtA/cdtB⁺(n=3), tcdA⁺tcdB⁺cdtA/cdtB^—(n=6), tcdA^—tcdB^—cdtA/cdtB^—(n=11). The multilocus sequence typing result showed that five ST types, including ST11 (n=3), ST3 (n=1), ST35 (n=2), ST36 (n=4), and ST109 (n=10). The phylogenetic tree results showed that all the isolates were clustered into two groups with 3 branches and 17 branches, respectively. [Conclusion] Clostridium difficile infections were found in pig herds in the main pig producing areas in Sichuan, and the molecular types of isolates were diverse. The main epidemic types were ST11, ST3, ST35, ST36 and ST109, and there was the risk of ST11 strain with high virulence.

Abstract:[Background] β-lactam antibiotics are commonly used drugs for the prevention and treatment of swine erysipelas, and penicillin (PG) is the preferred drug. [Objective] The use of transcriptomics and proteomics methods to preliminarily explore the mechanism of Erysipelothrix rhusiopathiae (E. rhusiopathiae) producing penicillin resistance, laying a foundation for further research on the resistance mechanism of E. rhusiopathiae. [Methods] The susceptibility to 26 antibiotics and the minimum inhibitory concentration (MIC) to PG, amoxicillin (AMX) and ampicillin (AMP) of the tested strains AErS, AEr51 and AEr31 were determined by the Kirby-Bauer disk diffusion method and the microdilution method; Then through transcriptomics sequencing (RNA-Seq) technology and tandem mass tag (TMT) quantitative proteomics technology to further explore the molecular mechanism of E. rhusiopathiae producing penicillin resistance. [Results] The resistance rates of AErS, AEr51 and AEr31 to 26 antibiotics were 34.62%, 34.62%, and 26.92%, respectively. AErS and AEr31 were sensitive to all β-lactams antibiotics, while AEr51 was sensitive to other β-lactams antibiotics except PG; the MICs of AErS, AEr51 and AEr31 to PG, AMX and AMP were 0.125, 0.500, 0.250 μg/mL, 32, 4, 2 μg/mL and 0.25, 0.50, 0.50 μg/mL, respectively; RNA-Seq analysis showed a total of 668 differential genes were screened in the AEr51/AErS comparison group, of which 434 were up-regulated and 234 were down-regulated. In the AEr51/AEr31 comparison group, a total of 403 differential genes were screened, of which 275 were up-regulated and 128 were down-regulated. The differentially expressed genes were mainly enriched in the metabolic pathway, ABC transport system, two-component signal transduction system, β-lactam resistance and other pathways, and the results of RT-qPCR verification were basically the same; TMT analysis showed that a total of 167 differential proteins were screened in the AEr51/AErS comparison group, among which 86 were up-regulated and 81 were down-regulated. A total of 159 differential proteins were screened in the AEr51/AEr31 comparison group, of which 80 were up-regulated and 79 were down-regulated. Differentially expressed proteins were significantly enriched in metabolic pathways related to microorganisms, amino acids, carbon, sulfur, and pyrimidine metabolism, and the results of PRM targeting verification were basically the same [Conclusion] ABC transport system, two-component signal transduction system, β-lactam resistance and other pathways played an important role in the development of E. rhusiopathiae resistance to PG, and were accompanied by life processes, such as microorganisms, amino acid, carbon, sulfur, and pyrimidine metabolism, etc.

Abstract:[Background] Tyrosinase is the key enzyme of melanin biosynthesis, which easily causes the pigment disorders and enzymatic browning of fruits and vegetables. At present, the development on tyrosinase inhibitor has obtained more and more attention. However some tyrosinase inhibitors have certain potential safety hazards, such as arbutin and kojic acid. Microorgansims are rich in resources and have many advantages. It has become a spot to search for tyrosinase inhibitors with strong specificity and high efficiency from microorganisms. [Objective] We investigated the effects of metabolite extracts of Salinicoccus ventosaetal strains B2-3-5 and B6-1-4 isolated from dabancheng Salt Lake of Urumqi in Xinjiang on tyrosinase activity. And also compared metabolic differences between the two strains during the fermentation. The aim of this study was to understand the inhibitory mechanism of tyrosinase activity of the strain B2-3-5. [Methods] Taken kojic acid as positive control, we determined the inhibitory effects of metabolite extracts produced by the two strains B2-3-5 and B6-1-4 on mushroom tyrosinase, respectively. We detected all the metabolites of the two strains under the same fermentation conditions by liquid chromatograph-mass spectrometer. We identified metabolic differences with univariate and multivariate analysis, Orthogonal Partial Least Squares-Discrimination Analysis (OPLS-DA) method. We carried out cluster analysis on the identified differential metabolites by Hierarchial Cluster Analysis (HCA) method and searched the differential metabolic pathways through the databases kyoto encyclopedia of genes and genomes (KEGG). [Results] These findings indicated that the inhibitory effects of metabolite extracts of the strain B2-3-5 on the diphenolase activity of mushroom tyrosinase was 67% and its IC₅₀ was estimated as 0.277 mg/mL, while the metabolite extracts of the strain B6-1-4 among the same genus had no inhibitory activity. We obtained 63 differential metabolites from the two strains by metabolomics methods. The types and relative contents of amino acids, vitamins and carboxylic acids were significantly higher in B2-3-5 than those in B6-1-4. The results of metabolic pathway analysis showed that these differential metabolites were mainly participated in 15 metabolic pathways, among which vitamin B6 biosynthesis pathway had the highest correlation with differential metabolites and fermentation process. [Conclusion] We speculated that the strain B2-3-5 might increase the contents of some small molecular compounds such as amino acids, vitamins and carboxylic acids, etc. to inhibit the tyrosinase activity. The upregulation of vitamin B6 biosynthesis pathway also suggested that the strain had the inhibitory activity of tyrosinase through producing vitamin B6 to interact with the essential amino group in tyrosinase or scavenging the reactive oxygen species (ROS) during the enzymatic catalytic process.

Abstract:[Background] Considering the emergence of "super bacteria" due to the abuse of antibiotics, the search for new antibacterial drugs is necessary for effectively conquering the problem of bacterial resistance. Since most antibacterial drugs are found from microorganisms, the research on endophytic fungi in medicinal plants can expand the medicinal resources, so it has a promising future for application. [Objective] To isolate the endomycete from the Cassia tora Linn. collected from Lushan Botanical Garden of Jiujiang, Jiangxi Province; screen out the antagonistic strains; isolate the secondary metabolites of such strains and analyze the physical and chemical properties of their antibacterial substances to prepare for the research of new antibacterial substances. [Methods] The antagonistic strains were screened by the cup-plate method and then identified based on morphological characteristics and molecular biology. Their secondary metabolites were isolated by silica gel, Sephadex LH20 and RP-C18 column chromatography, and the molecular weight and molecular formula of antibacterial substances were analyzed by liquid chromatography-mass spectrometry (GCMS) and EDS spectrometer. [Results] Penicillium citrinum ZH-11, a broad-spectrum antagonistic strain was screened out. As shown by the cup-plate method, it had a good bacteriostatic effect on eight indicator bacteria such as Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, Xanthomonas oryzae, Bacillus subtilis, Bacillus licheniformis, Candida albicans and Bacillus cereus etc. Active pure compound Y3 was isolated from the secondary metabolite of endomycete Penicillium citrinum ZH-11 with a molecular weight of 410.169 1 and a formula of C₂₄H₂₆O₆. When the concentration of Y3 was 10 µg/mL, the diameter of the inhibition zone against Escherichia coli and Bacillus thuringiensis was 16.23 mm and 17.27 mm, respectively. [Conclusion] This active substance is different from the known antibacterial active substances derived from Penicillium citrinum. The results laid a foundation for further exploration of the active products of Penicillium citrinum, and expanded our recognition of the endophytic fungi in Cassia tora Linn.

Abstract:[Background] The imbalance of human energy homeostasis is characterized by underweight, overweight and obesity. Gut microbiota is related to the maintenance of human energy homeostasis, but the feature of gut microbiota in different body mass index (BMI) populations need to be further explored. [Objective] To explore the feature and the co-occurrence network of gut microbiota of four BMI groups based on the American gut project (AGP), and to provide new theoretical basis for intervention of obesity and underweight based on gut microbiota. [Methods] Screen the gut microbiota samples with BMI information from AGP dataset, and divide the screened samples into four categories according to the BMI classification standards set by the World Health Organization:underweight (BMI<18.5 kg/m²), normal weight (18.52), overweight (252), obesity (BMI>30 kg/m²). By calculating and comparing the alpha diversity and beta diversity of gut microbiota, the overall feature and differences of gut microbiota in four BMI groups were explored. Use Microbiome Multivariable Association with Linear Models (MaAsLin) to analyze the correlation between different BMI groups and gut microbiota, and add geographical, age, and gender factors as confounding factors to the model for correction. SparCC was used to calculate the correlation of gut microbiota among the four BMI groups, and the gut microbiota co-occurrence network of four groups was constructed. [Results] The decreasing trend of alpha diversity in the underweight, overweight and obese population compared to the normal weight population was observed after the Wilcoxon-rank-sum analysis. As for the beta diversity, the significant difference of the structure of gut microbiota between the four BMI groups was found. There was no significant difference in the relative contents of Firmicutes and Bacteroidetes. Through MaAsLin analysis, and adding geographical, age, and gender factors as confounding factors to the model for correction, a total of 49 bacteria that were significantly related to BMI types were obtained. The topological structure of the co-occurrence network of the four groups of BMI populations were different. The networks of underweight and normal-weight population were more complex, while the networks of overweight and obese populations were less complex. [Conclusion] There were differences in the diversity, overall feature and co-occurrence network of the gut microbiota among the four BMI groups.

Abstract:[Background] Volvopluteus, a member of Pluteaceae, has only four species in the world currently. [Objective] To investigate the resources of macrofungi in northeast China. [Methods] Samples of macrofungi were collected, their morphology was observed and described in detail. Their DNA was extracted, rDNA ITS sequences were determined, and the molecular phylogenetic tree was constructed based on maximum likelihood and Bayesian inference. [Results] Among the specimens collected from 2019 to 2020 in Dunhua city, Yanbian Korean Autonomous Prefecture, Jilin Province, six V. michiganensis were found, which had not been found in China before. In the phylogenetic analysis, V. michiganensis collected from China was grouped into a branch with the type specimen. [Conclusion] V. michiganensis is a new record species in China.

Abstract:Disease is one of the important factors that affect the yield and quality of edible mushroom. At present, there is no intuitive understanding of the research status and development direction on mushroom diseases. Based on the articles focusing on edible mushroom disease published both domestic and overseas since 2010, the article counts, journals, citation rate, research institutions and keywords were analyzed, respectively. The co-occurrence analysis of key words was further carried out. Subsequently, the research focuses of edible mushroom disease were summarized. The analysis shows that green mold disease, brown blotch disease, cobweb disease, virus infection, wet bubble disease and soft rot disease are the six most concerned edible mushroom diseases both domestic and oversea. Additionally, the attention on dry bubble disease is decreasing, and the mushroom types infected by cobweb disease are increasing continuously. This paper also investigated the occurrence dynamics of edible mushroom diseases based on the historical trends, and considered that the detection of environmental harmful microorganisms, screening of disinfectants and control of soil-borne fungal diseases will be the key research fields in the future.

Abstract:Ectomycorrhizal fungi (ECMF) often form symbiotic relationships with many higher plants, and widely exist in nature. They play an important role in promoting the growth and nutrient absorption of plant, enhancing the stress resistance of the host and maintaining the stability of the ecosystem in forest. In addition to being closely related to host plants, ECMF often establish the symbiotic relationship with endobacteria, and interact physically and metabolically with bacterial communities during their life cycle. These bacteria had positive effects for ECMF on the growth of mycelia, increase of biomass and the formation of fruiting bodies. The article summarized the discovery of symbiosis, establishment of the symbiotic relationship, the promotion of growth, development of ECMF promoted by endobacteria, and the research methods of ECMF and endobacteria. In order to better consolidate the basic knowledge of biology and ecology of ECMF, and utilize the bacterial-fungal interactions to provide ideas for the biological control of edible ECMF, study of bacterial fertilizer, artificial domestication and cultivation.

Abstract:Soil-related evidence has been used for decades in forensic science. Forensic soil comparison test is mainly based on its physical and chemical properties, such as soil appearance, color, elemental composition, mineralogical properties and soil inclusions. Recently, with the development of high-throughput sequencing technology, independent of the traditional culture techniques, microbial communities could be directly tested and analyzed through all its DNA information in the soil. Then we use this information for sample comparison or source speculation in mock or real case. This highlights the great value and potential application of soil microbial evidence in case investigation and justice. This review summarizes the recent domestic and foreign research progress in forensic soil microbes, points out the potential applications for soil microbial diversity test in the field of forensic science, analyzes the influencing factors of soil microbial community diversity, and finally discusses the existing problems and future development direction of forensic soil microbiology.

Abstract:Due to the unique geographical environment and climate characteristics, the alpine grassland ecosystem is sensitive to grazing perturbation, contributing greatly to global greenhouse gases (GHGs) emissions. Therefore, it is significant to decipher the effect of grazing on GHGs emissions in alpine grassland. This study summarized the switch of GHGs sources or sinks and the response of soil microenvironment and microbial community structure under different grazing methods in alpine grassland. It is concluded that alpine grassland is the major CO₂ source, CH₄ sink, and N₂O source. Besides, grazing directly affects the aboveground plant, soil structure, temperature, humidity, and nutrient contents through selective feeding, trampling, and excreta returning of livestock, and then indirectly affects soil microorganisms and GHGs fluxes. This review provides a novel perspective for the healthy development and management of the alpine grassland ecosystem, the mitigation of global climate change in the alpine grassland.

Abstract:Nitrite-dependent anaerobic methane oxidation (N-DAMO) is a key link that couples the nitrogen cycle and the carbon cycle. It is mediated by Candidatus Methylomirabilis oxyfera. The discovery of the N-DAMO process has important implications for the study of the global carbon and nitrogen cycling. Through literature review, this article firstly summarized the influencing factors of N-DAMO reaction at home and abroad, N-DAMO reaction under different natural ecosystems. Then, it elaborated the functional structure, physiological and biochemical characteristics of N-DAMO bacteria, the enrichment and culture optimization of N-DAMO bacteria, and research techniques for N-DAMO reaction. Finally, the application research of N-DAMO was discussed. This article not only helps to reveal the coupling mechanism of global carbon and nitrogen cycles, but also provides a theoretical basis for N-DAMO reaction coupled with other anaerobic biological treatment processes to be applied to wastewater carbon and nitrogen removal.

Abstract:With the proportion of hair loss among young people increasing with years, hair health issues are of increasing concern. Hair loss affects people's quality of life and has a huge impact on psychological and social life. In recent years, research on the physiological functions of the gut microbiota is no longer limited to the gastrointestinal tract. Studies have shown that the intestinal tract and gut microbiota is closely related to the skin, suggesting the existence of the "gut-skin axis". In this article, based on the existing research on the "gut-skin axis", we summarize the recent literature to explore the possible links and potential mechanisms between the gut microbiota and hair loss, providing new insights and perspectives on the pathogenesis and therapeutic targets of hair loss.

Abstract:Actinomycetes can produce a rich diversity of small molecule compounds, but most of the actinomycetes have not been isolated and cultured because they are in a "viable but unculturable" state. One of the causes of "viable but unculturable" may be due to some environmental stresses, such as organic matter, heavy metal, antibiotics. These environmental factors make the cell form a dormant state, until they meet the suitable conditions for recovery growth. Resuscitation-promoting factor (Rpf) is a type of protein secreted by some actinomycetes. It was first discovered in Micrococcus luteus. Since then, more attention has been paid to the function and distribution of Rpf protein. Rpf protein can promote the recovery of some dormant Gram-positive bacteria and provide the possibility for "viable but unculturable" actinomycetes to be cultured in the laboratory. At the same time, the development of Rpf protein inhibitors for some pathogenic actinomycetes also provides a new way for the treatment of related diseases. Based on this premise, the structural composition, characteristics of the function, mechanism of action, and application prospect of Rpf protein were briefly reviewed.

Abstract:Hydrogenases are classified into tree types including[NiFe]-,[FeFe]- and[Fe]-hydrogenase depending on the different metals in the active center.[NiFe]- and[FeFe]-hydrogenases were found in various bacteria, while[Fe]-hydrogenase was only found in hydrogenotrophic methanogens. It catalyzes the hetero cleavage of H₂ to reduce the methenyl group of C1 carrier tetrahydrobiopterin into methylene group. Different from other two types of hydrogenases,[Fe]-hydrogenases do not contain electron-transmitting iron-sulfur clusters and bimetallic active centers. Additionally, the highly substituted pyridine ring and the Fe-acyl carbon bond in active center of[Fe]-hydrogenase indicated that there might be a new catalytic mechanism in hydrogenase. It also proposed that methanogens could synthesis this unique structural cofactor in a special way. This review summarizes the research progress of[Fe]-hydrogenase since its discovery in 1990, including physiological function, structural characteristics, catalytic mechanism, maturation process, and industrial application in future.

Abstract:Coronaviruses (CoVs) are a group of positive-sense, single-stranded RNA viruses with the largest genome, most of which can spread across species and infect humans. Some of the pathogens in the group are causing major public health problems and seriously threatening human health. The full-length genome of the viruses is about 25—31 kb in length, encoding multiple nonstructural, structural proteins (S, E, M, and N) and accessory proteins. For most coronaviruses, their accessory proteins are not indispensable for viral replication, but they are often involved in pathogenesis in hosts and act as functional proteins. These accessory protein genes are located at the 3' end of the viral genomes. Expression of these genes can be regulated at transcription level by the transcription regulating sequence (TRS) which locates at the beginning of the genes or at translation level by the codon usage bias of the protein-coding sequences. The accessory proteins belong to trans-membrane protein and carry unique protein transport motifs. These characters play decisive role for the formation of unique topological structures and intracellular transport of the proteins, thus directly affect their functions. A summary of the latest classification and genome structure of coronaviruses was made in the beginning of the article; then roles, categorization, protein transport motifs, topological structures and codon usage bias of the accessory proteins were discussed individually and prospects of research in the field were foreseen as well, aiming to help understand the biological characteristics of this category of proteins.

Abstract:Microbiology is not only a significant branch of life science but also an important basic course for Biology, Food science, Clinical Medicine in university education. The course is comprehensive with a wide range of knowledge, so how to mobilize students' interest in learning effectively will affect the classroom effect directly. In order to achieve good teaching effectiveness, teachers could use a variety of ways to improve students' interest and learning quality in the process of teaching. In this article, we make teaching design by the "interesting teaching method", and make teaching reform for the same major's classes of three different grades in a row. The teaching achievements are verified according to the students' final evaluation, process assessment (including attendance rate, classroom participation, attention concentration and so on) and students' feedback comments. As the results, in the final evaluation, the failure rate from the students of the classes with "interesting teaching method" is lower and the proportions of "good" and "excellent" students are higher than which students without "interesting teaching method" class. Otherwise, the process assessment scores of teaching reformed classes are much higher than that without it, which indicates that the "interesting teaching method" reform mobilized the students' interest in learning effectively. Therefore, we think that teachers can properly lead interesting contents into teaching design and promote them during the Microbiology class, it will help to ameliorate teaching atmosphere, arouse the enthusiasm and initiative of students and improve teaching quality.