Abstract:[Objective] Explore the diversity of gut microbiota of Tadorna ferruginea and its relationship with associated environment.[Methods] The fresh fecal samples of Tadorna ferruginea were randomly collected from five habitats at the northern Tianshan Mountain in the autumn of 2017, V3-V4 region was amplified with bacterial 16S rDNA universal primer, and high-throughput sequencing was performed, and then the software and online tools including BLAST, USEARCH and QIIME were used to analyze the bacterial diversity and compare the differences among the samples from different habitats.[Results] A total of 408 036 valid sequences and 1 014 OTUs were sequenced. The bacteria from the samples belonged to 25 phylum and 397 genera. At phylum level Proteobacteria and Firmicutes were dominant, meanwhile Bacteroidetes, Fusobacteria, Actinobacteria, Cyanobacteria, Spirochaetes, Saccharibacteria and Elusimicrobia are also abundance in the detected fecal samples. However, the proportions of these bacteria were diverse in these five habitat sites. In addition, the bacterial community of each site was significantly different at genus level. Cetobacterium, Megamonas, Pseudomonas, Lactobacillus and Streptococcus were the dominant bacteria in the samples from Aibi Lake, Anjihai reservoir, Kuitun reservoir, Moguhu reservoir and Bayi reservoir, respectively. Correlation analysis showed that the gut bacterial community was greatly influenced by the environment and food.[Conclusion] The diversity of gut microbiota of the Tadorna ferruginea in these five habitats in Xinjiang province was abundant, and there were some similarity and also specificity of the bacterial community in different sites, which were mainly related to the environment and food compositions in the habitat for Tadorna ferruginea.

Abstract:H5N8 subtype highly pathogenic avian influenza virus (HPAIV) has spread to Asia, Europe, Africa, the Americas, and other countries and regions with the migration of wild birds and commercial trade activities. H5N8 HPAIV caused two waves of pandemic in 2014-2015 and 2016-2019, and the third wave has broken out, leading to the death of a large number of poultry and wild birds and serious economic loss. Previous studies have shown that mutation can enhance the virulence of the virus to mammals such as canines, mice, and minks. The first human infection event was reported in February 2021, which is of important public health significance. This article reviewed the global epidemic situation of the H5N8 HPAIV in recent years, and discussed its origin and hazards. We suggest strengthening the active surveillance of AIV in wild birds, especially migratory birds, and performing early warning to reduce economic loss and threats to human health.

Abstract:Ferredoxins (Fds) are small proteins containing iron-sulfur clusters. They widely exist in nature and participate in biological physiological processes such as respiration, fermentation, nitrogen fixation, carbon dioxide fixation and hydrogen production, etc. Fd is particularly important for strictly anaerobic bacteria, because the energy metabolism of these organisms is highly dependent on biological components with low redox potential. Fd can flexibly adjust the redox potential by using their iron-sulfur centers to cater to the requirements of different redox potentials. Here, we take the Fd of anaerobic bacteria as examples and summarize related literatures about their structural types and physiological functions. Combined with the relevant work of our group, we dissect the possibility that Fd could produce reactive oxygen species (ROS). It may help to understand the importance of low-potential components such as Fd for anaerobes and could give clues and shed light on the question of intracellular ROS sources.

Abstract:There are complementary and competitive relationship among microorganisms in phycosphere for achieving nutrients, producing active substances and interactions. This leads to microbial symbiosis, competition, and cooperation under environmental stress, and toward to form a distinguished microbial ecological environment. The basic of such behaviors are the cell-to-cell communication among microorganisms, with signal molecules as the mediators. In this review, we systematically introduced the present researches relate to the phycosphere characteristics, cell-to-cell communication and the mechanism underlying signaling molecules transportation. Especially, we discussed the bottle-neck issues on understanding the trace signal molecules. We also verified the effects of gram-negative bacteria and microalgae phycosphere signal molecules on the regulation of microalgae gene expression pathways, based on the current research status of cell-to-cell communication in the phycosphere. Finally, research limitations on cell-to-cell communication in the phycosphere was summarized, and potential further studies were indicated. This work aimed to stimulate some ideas on the microbial ecology in phycosphere and microalgae biomass energy production.

Abstract:Bacterial biofilm is a structured community of microbial cells attached to biological or non-biological surfaces, which is composed of bacteria cells and their extracellular polymeric substances (EPS), including exopolysaccharides, protein, nucleic acid. Biofilms become one of the important causes of persistent infection, virulence and antimicrobial tolerance of pathogenic microorganisms. The biofilm matrix, as complex extracellular polymers, affect the structure and functional properties of biofilms. This article describes the composition and functions of two major categories of EPS, including cell surface components and extracellular components from some pathogenic bacteria. The physical and chemical properties of EPS matrixs and their roles in the biofilm virulence were mainly focused. Control strategies of biofilm matrix via multitargeted sites are also discussed. The aim of this review will provide theoretical support to explore biofilm forming mechanism and combating technologies in bacterial structured community.

Abstract:Treponema pallidum is the pathogen of the sexually transmitted disease syphilis that seriously endangers the physical and mental health of humans, and it is still difficult to achieve artificial culture in vitro. A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. This article describes the metabolic mechanisms of Treponema pallidum, such as nutrients transport, glycolysis pathways, and metabolite detours, in order to arouse the attention of researchers and further explore the physiological and metabolic functions of Treponema pallidum that are not yet understood, and break the bottleneck of Treponema pallidum in vitro artificial culture. To clarify the possible pathogenic mechanism of Treponema pallidum, to find new clinical treatment targets to provide reference.

Abstract:Bacillus subtilis was favored by researchers attributing to the excellent probiotic properties and strong stress resistance of spores. Owing to the special structure and unique physiological characteristics, the spores are an ideal place for foreign proteins such as enzymes and immunogens to anchor. B. subtilis spore surface display is one of the methods to express foreign proteins with high activity and stability. This article mainly reviewed the strategies and application prospects of displaying antigen proteins on the surface of B. subtilis spores to produce mucosal vaccines.

Abstract:[Objective] ω-hydroxy fatty acid (ω-HFAs) is a green, safe, non-toxic, ideal biodegradable material with good biocompatibility. It is widely used in chemical industry, food, pharmacy. Microbial fermentation, the method for producing ω-HFAs has important research significance and application prospects.[Methods] In order to obtain high-yield metabolically engineered strains of ω-hydroxy fatty acids, the POX gene in the β-oxidation of Candida tropicalis was continuously knocked out through homologous recombination technology. The effect of gene deletion on cell growth and the ability to synthesize ω-hydroxy fatty acids was investigated.[Results] The recombinant strain F4PT (ΔPOX4, ΔPOX5) was successfully constructed. Growth in the medium with fatty acid methyl esters (C₁₂~C₁₄) as the carbon source was significantly slower than that in glucose, indicating that the deletion of the POX4 and POX5 genes reduced the metabolic ability of fatty acid methyl esters. Secondly, through the fermentation experiment, the concentration of 12-hydroxydodecanoic acid and 14-hydroxytetradecanoic acid were 3.01 g/L and 8.69 g/L, the conversion rates were 11.44% and 33.14%, respectively. The ability to metabolize fourteen-carbon fatty acids is significantly stronger than that of twelve-carbon fatty acids.[Conclusion] Candida tropicalis with double deletion of FAO and POX genes can obviously accumulate ω-hydroxy fatty acids, but the accumulation efficiency of different carbon chains is different. The production and conversion rate of 14-hydroxytetradecanoic acid are higher, and the recombinant strain has the potential to convert methyl tetradecanoate to 14-hydroxytetradecanoic acid.

Abstract:[Objective] Wheat leaf rust caused by Puccina triticina (Pt) is one of the most damaging diseases in wheat production. The emergence of new races of Pt and the rise of inferior races led to the continuous overcoming of the resistance of varieties. Therefore, the analysis of deferentially expressed genes of Pt in different infection stages is of great significance to reveal the pathogenic molecular mechanism of wheat leaf rust.[Methods] In this study, deferentially expression genes in the interaction of pathogenic type THTT of Pt and the susceptible wheat (MU19) at the early interaction (inoculated after 6 h, 12 h, 24 h) and a later stage (6 d after inoculation) were analyzed with the RNA-seq transcriptome analysis method. Taking the early stage (MIQ) as the control group, and 6 d of affinity interaction (MI6d) as the experimental group to analysis the differentially expressed genes.[Results] 19 275 unigenes were up-regulated and 4 548 genes were down-regulated at 6 d. GO enrichment analysis found that the function of the deferentially expressed unigenes were mainly involved in metabolic processes of catalytic activities, cellular formation processes, single-tissue synthesis, intracellular organelle formation, nucleic acid binding and hydrolase activities, nitrogen compound metabolism, cellular metabolic process, etc. KEGG analysis showed that differentially expressed genes were involved in 109 pathways. The SNARE interaction pathway, porphyrin and chlorophyll metabolism pathway and ABC transporter pathway in vesicle transport were found to be related with pathogenicity of the Pt. Five genes were randomly selected from these three pathways for qRT-PCR analysis, and their expression trends were shown consistent with the results of transcriptome expression profile.[Conclusion] The function of the differently expressed genes at the early stages of inoculation may be related to the pathogenicity of the pathogen, while most of the differently expressed genes in the later stage are related to the growth and metabolism in the pathogen. There are obvious differences between the genes expressed at early and later stages. These results are important and provide fundamental references for future studies on the pathogenic mechanism of wheat leaf rust.

Abstract:[Objective] To study the separation, purification and biological activity of secondary metabolites from Monascus purpureus Mp-21. [Methods] The secondary metabolites of Monascus purpureus Mp-21 were isolated and purified using silica gel column, RPC₁₈ column and Sephadex LH-20 gel column and other column chromatographic separation techniques, and the structure of the compound was identified by spectroscopy technique (NMR and HR-ESI-MS). The anti-oxidation and hypoglycemic-related enzyme inhibitory activities of the identified compounds were determined. [Results] Ten active compounds were identified from the secondary metabolites of Monascus purpureus Mp-21, namely FMOC-ALLO-THR(TBU)-OH (1), quercetin (2), hesperetin (3), monascin (4), monasphilone A (5), oleanolic acid (6), β-sitosterol (7), ergosterol (8)、indole-3-carboxylic acid (9) and chlorogenic acid (10). Among them, compound 1 is a new natural product, which complements the information of the compound in nature and the information of the organic spectrum. Compounds 2, 6, 9 and 10 are discovered from the Monascus for the first time. In the in vitro antioxidant activity tests of O^2-, DPPH and OH^- free radicals, compound 2 (IC₅₀ of 5.07, 4.84 and 27.39 μg/mL, respectively) showed strong antioxidant capacity. Electron paramagnetic resonance (EPR) showed that compound 2 had strong scavenging ability on DPPH and OH^-. Compound 6 had strong inhibitory activity on α-glucosidase with IC₅₀ of 22.87 μg/mL. [Conclusion] Monascus purpureus Mp-21 is a high-quality germplasm resource with high development performance, and has the potential to be developed into functional food such as antioxidant and hypoglycemic.

Abstract:Ammonia oxidizing microorganisms mediate the oxidation of ammonium nitrogen in the soil, which is the first step of nitrification.[Objective] In the karst areas affected by large tunnel projects, understanding the response of ammonia oxidizing microorganisms to changes in soil moisture and nutrient condition has great significance for researching the changes of the ecological environment and the nitrogen cycle process caused by tunnel construction.[Methods] This study took four land uses (Grass land; bamboo land; mixed forest; vegetable land) of Longfeng trough valley, which is tunnel-affected-area and the Longche trough valley, which is non-tunnel-affected-area in Zhongliang Mountain, Beibei, Chongqing as an example, comparing the abundance of three ammonia oxidizing microorganisms (Ammonia oxidizing bacteria AOB, ammonia oxidizing archaea AOA, nitrite oxidizing bacteria CMX) and combining with the changes of soil water content, pH, and soil nutrient elements, to analyze the changes of ammonia oxidizing microorganisms caused by tunnel construction and its process mechanism.[Results] The results shows:① As the tunnel excavation exposed the underground aquifer, the level of groundwater decreased, the soil moisture content decreased, the pH and the nitrate nitrogen increased. The abundances of AOA, AOB and CMX of the tunnel-affected-area were significantly lower than the non-tunnel-affected zone, the abundances of the latter is 4.8, 4.4 and 3.9 times of the former respectively; ② Affected by the alkaline environment in soil and groundwater of karst area, as well as the vulnerability to leaking of solubles extremely easily, the concentration of substrates such as ammonium nitrogen is not the main influencing factors of ammonia oxidation microorganisms, the abundance of AOA is positively correlated with soil moisture content and soil buffering capacity (P<0.01), the abundances of CMX and AOB are both negatively correlated with soil nitrate nitrogen content (P<0.05), AOB abundance is also negatively correlated with soil pH (P<0.05).[Conclusion] This study reveals that soil physical and chemical properties like the soil moisture content and pH in karst areas are the main factors affecting the abundance of three ammonia oxidizing microorganisms. The leakage of groundwater and the decrease of soil effective moisture caused by tunnel construction are responsible for the decrease of ammonia oxidizing microorganisms abundances, which also change the nitrification process in tunnel-affected-area to some extent. However, the degree of influence and the changes of nitrogen cycle which more microorganisms involve in need to be further studied in detail.

Abstract:[Objective] Ascoviruses are humor transmitted insect viruses that have unique pathogenicity and are competent to develop into bioinsecticides. Heliothis virescens ascovirus 3h (HvAV-3h) is the first ascovirus strain that isolated in China, and the 3H-117 protein encoded by HvAV-3h are reported as a structural protein of HvAV-3h.[Methods] In order to further illustrate the function of 3h-117, the Bac-to-Bac insect expression system was employed to construct a recombinant Autographa californica multiple nuclepolyhedrovirus (AcMNPV) by inserting 3h-117 into the genome of AcMNPV.[Results] Compared with the wild type control AcMNPV (AcMNPV-Egfp), the generated recombinant baculoviurs (AcMNPV-117) had a significantly reduced budded virus production and viral DNA copies in the infected Sf9 cells during 72-96 hpi, and an enlarged occlusion bodies was found in AcMNPV-117. Further bioassays showed that the 90% lethal dosage of AcMNPV-117 against the third instar Spodoptera exigua are significantly higher than the 90% lethal dosage of AcMNPV-Egfp. Furthermore, the growth of the tested S. eixuga larvae infected with AcMNPVs was slower than that of the healthy larval growth, and the average daily food intake of the tested larvae was also inhibited by the baculovirus infection. The confocal observation showed that 3H-117 is associated with the nuclear actin polymerization in AcMNPV-117 infected Sf9 cells, which indicated that the function of 3H-117 in the infectious procedures of ascovirus is associated to the disintegration of host cell nucleus.[Conclusion] The results obtained in this study confirmed the function of 3h-117, which laid a foundation for the molecular biology research of ascovirus.

Abstract:[Objective] Our main goal was to clarify the adaptation mechanism of Brevibacterium species to the marine environment.[Methods] In this paper, six Brevibacterium strains isolated from different sea regions and belonging to different operational taxonomic units were sequenced, assembled and annotated. Combined with the genomic data of 23 typical and atypical Brevibacterium strains downloaded from NCBI, pan genomics analysis and species evolution analysis were carried out.[Results] Pan genomics analysis showed that the Brevibacterium strains had an open pan genome, which was consistent with the diversity of its living environment. There are obvious differences between marine Brevibacterium strains and other habitat Brevibacterium strains at the genomic level, mainly in the aspects of gene family expansion and contraction, transporter family, metabolic pathway and CRISPR.[Conclusion] These differences at the genomic level preliminarily revealed the adaptability of marine Brevibacterium strains to marine environment, which laid the foundation for a deep-going understanding of the environmental adaptation mechanism of Brevibacterium strains.

Abstract:The core symbiotic bacteria of insect vectors have the potential to be used as genetically engineered bacteria to control arboviruses. Laodelphax striatellus is an important agricultural pest and also the transmission vector of rice stripe virus, the causative agent of rice stripe disease that occurred with dramatic yield reduction or even no harvest.[Objective] In this study, the bacterial communities of L. striatellus were sequenced through 16S rDNA next-generation sequencing methods, and the core symbiotic bacteria were identified.[Methods] From 2018 to 2020, L. striatellus have been collected from the rice fields in Kunming of Yunnan Province, and in Kaifeng and Fanxian of Henan province, as well as in the greenhouse, in which microbiomes were sequenced and analyzed.[Results] All 37 L. striatellus sequencing samples carry both intracellular and extracellular bacteria. In the genus level, the intracellular bacteria Wolbachia exhibited the highest average relative abundance of 51.96%. Its representative OTU (OTU45) accounts for 96.55% of the total number of 85 OTUs and exists in all sequenced insect samples. Except for Wolbachia, all the other common core bacteria were extracellular, in which the four core genera with high abundances were Acinetobacter, Pantoea, Enterobacter and Pseudomonas, with average relative abundances of 0.25%-2.97% in all ten common core genera. There are dominant bacteria in insect samples from different rice fields. High-abundant Rickettsia were found in Kunming samples, both Arsonophonus and Cardinium were identified in Fanxian samples, and Acetobacter, Sulcia and Pantoea were found in Kaifeng samples. These rice-field dominant bacteria include a variety of intracellular bacteria. When the rice-field insects were fed with sucrose for 24 h in the greenhouse, the relative abundance of Wolbachia increased significantly and the microbial composition became closer to that of the greenhouse. Within the offsprings of these rice-field insects, the intracellular bacteria, except for Wolbachia, lost or existed in a few samples with low level.[Conclusion] This study preliminarily clarified the symbiotic bacterial composition of L. striatellus, identified common core bacteria and identified dominant symbiotic bacteria in the three Chinese rice areas, providing engineered bacteria for antiviral research through insect vector's core bacteria.

Abstract:[Objective] In order to investigate the influence of Mn²⁺ on the removal efficiency and microbial community diversity of A/O-BAF system, the removal efficiency of Anoxic and Aerobic Biological Aerated Filter were analyzed under different concentration of Mn²⁺ at 15.[Methods] Manganese ions were added into the influent water to investigate the change of reactor treatment efficiency under the temperature of 15, hydraulic load 0.50 m³/(m²·h), gas water conditions than 10:1. The BAF biofilm samples were analyzed by high-throughput sequencing.[Results] The results showed that the removal efficiency of COD, NH₄⁺-N, TN and TP in Anoxic and Aerobic Biological Aerated Filter under concentration of 2.0 mg/L Mn²⁺ were increased by 3.51%, 2.21%, 6.26% and 12.13%. Under the concentration of 4.0 mg/L Mn²⁺, the removal efficiency of COD, NH₄⁺-N, TN and TP were increased by 4.24%, 1.92%, 7.75% and 10.73%, respectively. The number of denitrifying bacteria and Nitrobacteria increased significantly, and the number of Nitrosomonas decreased. The number and activity of biofilms also increased. The results of high-throughput sequencing showed that the community diversity increased significantly under 2.0 mg/L and 4.0 mg/L Mn²⁺, and the number of OTU samples increased from 4 430 to 5 659 and 5 556. The relative abundance of Proteobacteria, Firmicutes, Xanthomonas, Rhodocyclaceae, Comamonadacea, Comamonas, Aedonia, Denitratisoma, Sulfuritalea and Thermomonas increased in BAF. The relative abundance of some microorganisms, such as Denitratisoma, Sulfuritalea, and Thermomonas, decreases with higher concentrations of manganese ions. The relative abundance of Nitrosospira and Nitrospirae decreased under the action of manganese ions.[Conclusion] Manganese ion can improve the removal effect of pollutants in the Anoxic and Aerobic Biological Aerated Filter mainly by promoting the nitrification and denitrification and the growth of phosphorus removing microorganisms at 15, but the promotion effect on nitrification is not obvious. Nitrosomonas are more susceptible to the toxic effects of manganese ions.

Abstract:[Objective] Lytic polysaccharide monooxygenase (LPMO) is a recently discovered copper ion-dependent oxidase, which can break glycosidic bonds by oxidation, thus significantly improving the efficiency of polysaccharides degradation. However, it is easy to be inactivated and difficult to evaluate the activity of LPMO due to the properties of its substrates and the diversity of its released products.[Methods] In this study, we established a spectrophotometric activity assay to detect the chitin-active LPMO (BtLPMO10A) using 2,6-dimethoxyphenol (2,6-DMP) and H₂O₂ as substrates, and to evaluate the stability of LPMO during chitin degradation.[Results] The results suggested that high concentration of enzyme, 2,6-DMP or H₂O₂ would deviate the reaction from linear range. The K_m of BtLPMO10A toward 2,6-DMP and H₂O₂ was determined to be 0.53 mmol/L and 5.31 mmol/L respectively. It suggested BtLPMO10A possessed a higher affinity toward 2,6-DMP and H₂O₂ than NcLPMO9C. BtLPMO10A was easy to be inactivated in the presence of reducing agent ascorbic acid. The substrate chitin could stabilize the enzyme, but the activity still decreased during the degradation of chitin.[Conclusion] This work evaluated the factors that impacted on the assay for detecting the activity of BtLPMO10A using 2,6-DMP as substrate, and estimated the stability of BtLPMO10A during chitin degradation. It will provide important information for the investigation of chitin-active LPMOs.

Abstract:[Objective] To screen and identify microorganisms capable of metabolizing polyethylene terephthalate monomer terephthalate and analyze the catabolic pathways.[Methods] Collected samples from Qingdao Xiaojianxi solid waste comprehensive disposal plant and screened strains capable of metabolizing terephthalate using the medium with terephthalate as the sole carbon source; 16S rRNA phylogenetic tree analysis was used to determine the taxonomic status of TPA3 strain; de novo sequencing was implemented using the second-generation and the third-generation high-throughput sequencing technologies; the terephthalate and ethylene glycol catabolic pathways and the related genes of TPA3 were analyzed through amino acid sequence alignment; the genetic manipulability of TPA3 was verified by the reported genetic manipulative techniques.[Results] TPA3 was identified as Pseudomonas stutzeri; it can metabolize 10.60 g/L terephthalate within 30 hours at 30℃. After culture and domestication, it could also catabolize ethylene glycol. The complete genome was composed of one chromosome and three plasmids for a total genome size of 4.55 Mb. It was speculated that TPA3 strain catabolized terephthalate in a classic way, and the ethylene glycol catabolic pathway should be similar to Pseudomonas putida KT2440. The genetic manipulation technology of Pseudomonas could be used for TPA3.[Conclusion] TPA3 strain could degrade polyethylene terephthalate monomers terephthalate and ethylene glycol, and could be genetically modified, showing potential application value in polyethylene terephthalate waste biological treatment technology.

Abstract:[Objective] To investigate the interaction mechanism between endophytic bacteria and host poplar, the diversity, structural characteristics and biological functions of the endophytic bacterial community in the leaves and trunk of Populus euphratica were analyzed.[Methods] By using Illumina MiSeq high-throughput sequencing technology, we performed Alpha and Beta diversity analysis, community composition analysis, and functional prediction compared with metabolic database of the endophytic bacteria on three groups of Populus euphratica leaves and three groups of trunk samples collected from poplar forests in Kashgar, Xinjiang, China. And endophytic bacteria from the samples were isolated and purified.[Results] The dominant groups of Populus euphratica leaves were Proteobacteria (40.71%), Actinobacteria (21.76%), Bacteroidetes (14.24%), and Firmicutes (13.92%), and the dominant groups of Populus euphratica trunk samples were Firmicutes (56.91%), Actinobacteria (37.01%). The results of PCoA analysis showed that there were significant differences in the endophytic bacterial community structure between Populus euphratica leaves and trunks, and the community structure of the same type of tissue samples was similar. Functional prediction revealed that the metabolic pathways and enzymes with higher abundance of endophytes were mostly related to cell wall and cell membrane, efflux pumps, oxidative stress, compatible solutes, energy metabolism, etc. A total of 44 cultivable endophytic bacteria belonging to five genera were isolated, among which Bacillus accounted for the largest proportion.[Conclusion] The diversity and abundance of endophytic bacteria in Populus euphratica leaves were higher than in trunk tissues, and different Populus euphratica tissues also influenced the composition of theendophytic bacterial community due to their different biological functions. The functions of the endophytic bacterial community corresponded to the host tissues, and the habitat in which Populus euphratica was located also influenced the functional expression of its endophytic bacterial community. The cultivable endophytic bacterial community of Populus euphratica has salt-tolerant and other characteristics, which are potential strain resources.

Abstract:[Objective] We evaluated the safety of Corallococcus sp. strain EGB to provide a scientific basis for the development of novel biocontrol agent and its safe application to the environment.[Methods] The genotoxicity of the cells and fermentation products of strain EGB were investigated by Ames test, polychromatic erythrocyte micronucleus test, and testis spermatocyte chromosome aberration assay in mice. Intragastric administration was employed to study the acute toxicity and 28-d subchronic toxicity.[Results] The results of Ames test, polychromatic erythrocyte micronucleus test, and testis spermatocyte chromosome aberration assay in mice revealed that the cells and fermentation products of strain EGB exhibited no genotoxic potential within the testing system. The LD₅₀ values of the cells and fermentation products of strain EGB were greater than 10 g/kg BW in ICR mice. In the 28 days subchronic toxicity test, the body weight, food and water consumption, blood biochemical indexes, blood routine parameters, organ weights, and histopathology of treatment groups showed no difference compared with those of the control group (P<0.05).[Conclusion] Strain EGB and its ferments could be classified as no toxicity and are safe for practical application in the plant disease control and bioconversion.

Abstract:[Objective] This study was aimed to investigate the effects of long-term phosphorus (P) supply on the diversity, structure, composition and network characteristics of bacterial community encoding alkaline phosphatase gene and their relationship with the forms of soil organic P, so as to provide scientific basis for exploring the community characteristics of organic P cycling functional microorganisms and improving the mineralization and utilization of soil organic P.[Methods] Based on the long-term P application experiment in calcareous soil of wheat-maize rotation system in North China Plain (started in 2008), the experiment included six P levels:0, 12.5, 25, 50, 100, 200 kg P/hm² (P0, P12.5, P25, P50, P100 and P200 respectively) and the molecular forms of organic P in soil were determined by nuclear magnetic resonance (NMR), high-throughput sequencing technology was used to analyze the bacterial community of phoD gene (encoding alkaline phosphatase), and explore the bacterial community characteristics of phoD gene and its relationship with the forms of organic P in soil.[Results] With the increase of P supply level, the concentration of phosphate monoester did not change significantly, but the concentration of phosphate diester increased significantly; the α-diversity of the phoD harboring bacterial community was first unchanged (P0 to P50) and then decreased (P50 to P200), and the community structure of phoD harboring bacteria changed significantly. The dominant taxa named Pseudomonas and Masslis at genus level decreased significantly, while Mitsuaria and Kribbella increased significantly with the increase of P supply level. Mitsuaria was positively correlated with the concentration of available P in soil, and negatively correlated with the activity of phosphatase. There was a significant negative correlation between the relative abundance of Pseudomonas and the concentration of total organic P in soil, and a significant positive correlation between the relative abundance of Lysobacter and the concentration of total organic P in soil. In the network analysis, the proportion of positive edges was the highest in P0 treatment, followed by P50 treatment, and the lowest in P100 treatment. In the treatments of P0, P12.5, P25 and P50, specific keystone taxa such as Bradyrhizobium, Stackbrandtia, Burkholderia, Bradyrhizobium and Lysobacter were significantly correlated with the concentration of organic P (including total organic P, phosphate monoester and phosphate diester), however, the keystone taxa under P100 and P200 treatments has no significant correlation with the forms of organic P.[Conclusion] The levels of P supply significantly affected the physicochemical properties of soil, such as pH, the forms and quantities of organic P, and then affected the changes of α-diversity, community structure, community composition, network characteristics and keystone taxa of phoD harboring bacterial community.

Abstract:[Objective] This study aimed to investigate the differences in the structure of fungal community in the feces of grazing Tibetan pigs, captive Tibetan pigs, and commercial pigs[Duroc×Landrace×Yorkshire (DLY) pigs, aged 5 months] in the Tibet Plateau and to obtain the fungi associated with the digestion of dietary crude fiber.[Methods] The apparent digestibility of dietary crude fiber of grazing Tibetan pigs, captive Tibetan pigs, and DLY pigs was determined via digestion experiments. The full-length ITS region of fecal fungi was determined by single molecule real-time sequencing technology to analyze the structure and diversity of fungal community. The Pearson correlation analysis was performed between apparent digestibility of dietary crude fiber and fungal community.[Results] A total of 58 fungal species belonging to 4 phyla, 13 classes, 23 orders, 39 families, and 55 genera were identified. At each taxonomic level, the fungal taxa in the feces of grazing Tibetan pigs were more than those of either captive Tibetan pigs or DLY pigs (P<0.05). Ascomycota and Basidiomycota were the dominant phyla, and the fungal abundance at the phylum level had no significant difference among the

grazing Tibetan pigs, captive Tibetan pigs, and DLY pigs (P ≥ 0.05). At the levels of class, order,

family, genus, and species, the fungal abundance of grazing Tibetan pigs was significantly higher than that of either captive Tibetan pigs or DLY pigs (P<0.05). The grazing Tibetan pigs had the most diverse fungi with unique operational taxonomic units (P<0.05). The principal coordinate analysis revealed that grazing Tibetan pigs had different fecal fungi compared with captive Tibetan pigs and DLY pigs (P<0.05). The grazing Tibetan pigs had higher apparent digestibility of dietary crude fiber than captive Tibetan pigs and DLY pigs (P<0.05). Pearson correlation analysis showed that Phialemonium atrogriseum, Phialemonium inflatum, and Podospora communis had a positive correlation with the apparent digestibility of dietary crude fiber (P<0.05).[Conclusion] The grazing Tibetan pigs had stronger fiber digestibility than the captive Tibetan pigs and DLY pigs. In the future, we could identify more fungal groups from their gut. This information would be helpful in studying the excellent characteristics of Tibetan pigs, such as their tolerance to roughage.

Abstract:[Objective] To study the effects of different concentrations of Zn(II) on the metabolic activities, especially the denitrification of the aerobic denitrifier Acinetobacter sp. JR-142.[Methods] An aerobic denitrifier was screened out and its growth conditions were optimized. The growth curve, denitrifying efficiency, and cell morphology of the strain were measured in the presence of Zn(II) at different concentrations. We measured the activities of cell characteristic enzymes (nitrate reductase and nitrite reductase) exposed to different concentrations of Zn(II), and then analyzed the relationship between napA & nirS expression levels and enzyme activities.[Results] An aerobic denitrifying strain was obtained and identified as Acinetobacter sp. JR-142. Under the conditions of sodium succinate as carbon source, C/N ratio of 6, pH 7.0, temperature at 30℃, shaking speed of 180 r/min, the aerobic denitrifier showed the highest activity. Zn(II) at the concentration of 3.25 mg/L promoted the cell growth and aerobic denitrification, while that at the concentration higher than 52 mg/L showed inhibitory effects. The nitrate reductase and nitrite reductase activities in the control group and JR+0.05 treatment group were higher than those in the JR+0.8 treatment group. At the time point of 24 h, the relative expression levels of aerobic denitrifying genes napA and nirS in JR+0.05 treatment group were significantly higher than those in control group, which further indicated that 3.25 mg/L Zn(II) can promote aerobic denitrification. At the time points of 24 h and 32 h, the relative expression levels of the two genes in the control group and JR+0.05 treatment group were much higher than those in the JR+0.8 treatment group, which indicated that 52 mg/L Zn(II) can inhibit the reaction.[Conclusion] This study systematically analyzed the effects of Zn(II) at different concentrations on the growth and aerobic denitrification of Acinetobacter sp. JR-142 for the first time. It provides a data basis for the biological treatment of wastewater with nitrate-heavy metal combined pollution.

Abstract:[Objective] To assemble a complete pholipomycin biosynthetic gene cluster (pho-BGC) based on the nosokomycin B₂ biosynthetic gene cluster (noso-BGC) derived from Streptomyces lincolnensis NRRL2936, and activate pho-BGC through heterologous expression for production increase of pholipomycin using the screening of the chassis hosts.[Methods] Firstly, three genes moeA4, moeB4, and moeC4 were cloned from the genome of moenomycin producer Streptomyces ghanaensis ATCC 14672 and introduced into lincomycin gene cluster deletion strain JCK126, which yielded the recombinant strain LX19. The fermentation extract detection of LX19 showed that pho-BGC was silent in S. lincolnensis NRRL2936. Subsequently, the gene cassette carrying moeA4, moeB4, and moeC4 was inserted into noso-BGC by gene assembly, resulting in the plasmid pJQK572 containing the intact pho-BGC. Then, plasmid pJQK572 was introduced into Streptomyces coelicolor M1152, S. lividans SBT18, S. lividans LJ1018, and S. coelicolor M1446, which generated strains LX20, LX21, LX22, and LX23, respectively. Finally, the pholipomycin production ability of different recombinants was estimated by bioactivity analysis and UPLC-TOF MS of fermented extracts, and the chemical structure of pholipomycin was identified by ESI-MS².[Results] The complete pho-BGC achieved heterogeneous expression in S. coelicolor M1152. Moreover, the yield of pholipomycin was increased by 20% in S. coelicolor M1446 (carrying four copies of the ΦC31-attB sites).[Conclusion] This study determined that pho-BGC was silent in S. lincolnensis by systematic fermentation assay. Then, plasmid pJQK572 containing the complete pho-BGC was constructed based on noso-BGC. The liquid-mass spectrometry for the fermented extracts of different pho-BGC heterogeneous hosts determined that pholipomycin was successfully synthesized in S. coelicolor M1152. Moreover, the yield of pholipomycin in strain LX23 was increased by 20% through multi-copy integration of pho-BGC. The successful construction and heterologous expression of pho-BGC laid a foundation for revealing the biosynthesis mechanism and increasing the yield of pholipomycin.

Abstract:[Objective] This study compared the differentiation of endophytes in the intestines of Helicoverpa armigera under feeding normal diet and cottonseed meal diet, and studied gossypol tolerance and degradation effects of the screened strains, which provided theoretical and experimental support for further research on the detoxification of Helicoverpa armigera intestinal microorganisms in cotton-derived feed.[Methods] The gossypol tolerant endophytic bacteria were isolated in the intestinal tract of Helicoverpa armigera under different feeding conditions, selective culture of gossypol acetate single carbon source microorganism, and 16S rRNA gene sequence were analyzed and identified. The detoxification effect of the strains was analyzed by cultivated in a certain concentration of gossypol acetate liquid, and the compound ratio was used to study the effect of the compound bacteria on gossypol degradation.[Results] A total of 32 strains were isolated from Helicoverpa armigera, of which 17 strains were potential new taxon, and 14 were potential new species, and 3 were possible potential new genera. 17 strains were isolated from the intestinal tract of Helicoverpa armigera under feeding normal diet, belonging to 10 genera in 3 phyla of bacteria, 15 strains were isolated from the intestinal tract of Helicoverpa armigera under feeding cottonseed meal diet, belonging to 8 genera in 2 phyla of bacteria. With the increase of the concentration of gossypol acetate in the selective medium, the number of intestinal bacteria resistant to 100, 300, 500, 1 000 mg/kg gossypol acetate in the cottonseed meal feed groups were higher than those in the normal feed groups. Among all the tolerant strains, 15 strains had a degradation rate of more than 50%, and the highest degradation rate of gossypol was 90.83%. In the experiment of compound degradation of gossypol by intestinal bacteria, the degradation rate of gossypol in the experimental group were significantly higher than that in the control group (P<0.05), and the highest degradation rate of gossypol was 87.04%.[Conclusion] It was proved that there were some higher ability of degrading gossypol strains in the intestinal endophytic bacteria of Helicoverpa armigera, which enriches the resource of the strains degrading gossypol, and preliminary research on the potential of compound bacteria in the development and application of cotton-derived feed was conducted, which provided a new way for the biodegradation of gossypol in cotton-derived feed.

Abstract:[Objective] This study aims to investigate how mixed bacteria modulate the oviposition preference of Drosophila through competition and to explore the effect of bacterial balance on Drosophila offspring based on the survival rate and developmental duration.[Methods] Lactobacillus plantarum, Escherichia coli, and the mixture of the two (hereinafter referred to as the mixture) were coated on the casein-sucrose-agar medium (food of Drosophila), respectively. The egg-laying behavior of Drosophila was assayed by a two-choice device. Bacterial count was calculated with the plate method and the effect of the mixture on the survival of Drosophila was evaluated based on the survival rate of pupa.[Results] Drosophila avoided laying eggs on the food that was fermented by E. coli at 37℃, while it was attracted to lay eggs on the food fermented by L. plantarum at 37℃. The mixture (at the ratio of 1:1) significantly repelled the oviposition of Drosophila, as manifested by the oviposition index (OI) of only -0.46, suggesting Drosophila oviposition preference is an ideal mode for studying the balance of mixed bacteria. Bacterial counting showed that E. coli dominated the medium, with the count 4.43 folds that of L. plantarum, which caused the oviposition avoidance of Drosophila. As expected, increase in the ratio of L. plantarum in the mixed bacteria mitigated or even reversed the oviposition avoidance of Drosophila to fermented diet, indicating that the inoculation ratio of bacteria affected the balance of the flora. With L. plantarum and E. coli mixed at equal ratio, the OI of Drosophila was 0.41 at 25℃, -0.06 at 30℃, and -0.45 at 35℃, suggesting temperature impacted bacterial balance. The metabolites of L. plantarum, particularly lactic acid, inhibited the growth of E. coli, which contributed to the oviposition preference of Drosophila. The survival rate of pupa was 14% when E. coli dominated and 91% when L. plantarum was dominant, indicating that the growth balance of the mixture influenced the survival rate of Drosophila.[Conclusion] Bacterial ratio and culturing temperature determined the final oviposition selection of Drosophila and Drosophila adjusted the oviposition behavior based on bacterial balance to facilitate the survival of offspring.

Abstract:[Objective] This paper aims to investigate genes related to the production of S-equol from daidzein in Clostridium sp. C1, which is expected to serve as a reference for exploring the S-equol transformation mechanism in C1 and discover genes for the synthesis of S-equol by synthetic biology techniques.[Methods] Through the third-generation sequencing (GridION), assembly, and function annotation of the whole genome of C1, genes related to the biotransformation of S-equol were screened and identified.[Results] The genome was 3 035 113 bp, with 3 166 protein-coding genes, 53 tRNA genes, 15 rRNA genes, 4 ncRNA genes, and 1 genomic island. Bioinformatics analysis revealed that C1-07020 protein shared 44.8% amino acids and 3 conserved domains with daidzein reductase of Lactococcus sp. 20-92. In vitro verification showed that C1-07020 protein had similar functions to daidzein reductase. In addition, no other S-equol-producing gene clusters or other functional genes were identified from C1, which suggests that C1 may have a different metabolic mechanism from other S-equol-producing bacteria.[Conclusion] One S-equol-producing gene was identified from C1 and C1 might have a unique S-equol synthesis mechanism. The results of this study can serve as a reference for further exploring S-equol-producing genes, the production mechanism, and in vitro utilization of the genes.

Abstract:[Objective] Root-knot nematode is destructive and difficult to be controlled. Virgibacillus dokdonensis MCCC 1A00493 is antagonistic to Meloidogyne incognita in vitro. In this study, we isolated and identified the nematicidal compound in the fermentation broth of MCCC 1A00493 and explored the modes of action, aiming at laying a theoretical foundation for the effective control of plant pathogenic nematodes.[Methods] The nematicidal compound in the supernatant of MCCC 1A00493 fermentation broth was isolated and purified by silica gel column chromatography and semi-preparative high-performance liquid chromatography, and the structure of the purified product was identified by liquid chromatography-mass spectrometry and nuclear magnetic resonance. The repellent activity, trapping activity, fumigation activity, and egg hatching-inhibiting activity of the compound were tested.[Results] The compound was identified to be 4-vinylphenol, which had the contact activity, fumigation activity, egg hatching-inhibiting activity, repellent activity at high concentration, and trapping activity at low concentration against the nematodes. At 15 μg/mL, 4-vinylphenol demonstrated contact activity, as manifested by the high corrected death rate (71.23%±9.06%) of M. incognita after 72 h. At 20 mg/mL, it showed fumigation activity and the death rate was 100% after 24 h. Inhibition rate of M. incognita egg hatching reached 66.2% after 10 days of exposure to 100 μg/mL 4-vinylphenol. Nematodes were repelled by 10 mg/mL, but attracted by 1 mg/mL 4-vinylphenol on agar plates. In Pluronic gel, nematodes were repelled by 25 mg/mL, and attracted and killed by 5 and 10 mg/mL 4-vinylphenol.[Conclusion] The compound 4-vinylphenol, identified from V. dokdonensis, possessed multiple modes of action against M. incognita, especially the repellent effect at high concentration and trapping effect at low concentration. Thus, it can be used as a potential bionematicide.

Abstract:[Objective] Microsporidia are a group of obligate intracellular parasites that can infect humans and nearly all animals. Here, we studied the polar tube protein 4 of Encephalitozoon hellem (EhPTP4) on its subcellular localization and functions as a potential secretory virulence factor in host cells.[Methods] A polyclonal antibody against EhPTP4 was produced to verify the protein subcellular localization in E. hellem-infected cells using indirect immunofluorescence assay (IFA) and Western blotting. HEK293 cells were transfected with wild-type or mutant EhPTP4 fused with HA-EGFP, and the impacts on pathogen proliferation, protein subcellular localization and sequence functions were assessed. RNA sequencing of EhPTP4-transfected cells was conducted to identify differentially expressed genes (DEGs) and pathway responses. The regulatory effects of candidate DEGs were analyzed via RNAi and cell transfection, and the effects were determined with RT-qPCR and Western blotting.[Results] EhPTP4 contains a signal peptide at the N-terminal, a nuclear localization sequence (NLS) and a histidine-rich domain (HRD) at the C-terminal. In the infected and transfected cells, EhPTP4 was secreted into the host nucleus. Transfection and overexpression of EhPTP4 in HEK293 cells significantly promoted pathogen proliferation. RNA-seq of the transfected cells showed that genes involved in endoplasmic reticulum (ER)-associated degradation (ERAD), a quality control mechanism that allows for the targeted degradation of proteins in the ER, were prominently upregulated. Upregulation of the ERAD genes PDIA4, HERP, HSPA5 and Derlin3 determined by RNA-seq data was verified using RT-qPCR and Western blotting. Protein ubiquitination of the transfected cells was then assayed and found to be markedly increased, confirming the activation of ERAD. PDIA4 knockdown with RNAi significantly suppressed the expression of HERP, indicating that PDIA4 is vital for the modulation by EhPTP4. Moreover, EhPTP4^ΔHRD, a deletion mutant lacking the HRD, could not cause the upregulation of ERAD genes, indicating that the HRD is essential for the function of EhPTP4.[Conclusion] This study is the first report on a microsporidian secretory protein that targets the host nucleus to upregulate the ERAD pathway and subsequently promote protein ubiquitination. Our work provides new insights into microsporidia-host interactions.

Abstract:[Objective] Marine fungi are ideal producers of novel natural products. This paper aims to deepen the understanding of the diversity of culturable marine fungi and identify potential useful marine fungi.[Methods] Fungi were isolated from offshore and pelagic seawater by membrane filtration. Through the isolation and purification of strains and internal transcribed spacer (ITS) sequencing, the diversity of culturable fungi in neritic water, bathyal water, and abyssal water was analyzed. The solid plate method was used to screen strains with antibacterial activity.[Results] A total of 548 fungi were isolated and they fell into 44 species, 24 genera, 19 families, 11 orders, 6 classes, and 2 phyla, including 15 marine species and 3 novel taxa. At the class level, Dothideomycetes dominated (13 species), followed by Sordariomycetes (11 species) and Eurotiomycetes (11 species). At the genus level, Penicillium, Cladosporium, Exophiala, and Simplicillium were dominant. The 44 species presented 5 geographic distribution pattern:presence in neritic, bathyal, and abyssal areas (15 species), presence only in neritic area (5 species), absence in neritic area (12 species), absence in bathyal area (5 species), and absence in abyssal area (7 species). A total of 12 species had antibacterial activity. Among them, Hortaea werneckii showed the broadest antibacterial spectrum, and Simplicillium cylindrosporum demonstrated the strongest inhibition on Staphylococcus aureus MCCC1A0646.[Conclusion] This study reveals the diversity and distribution pattern of culturable fungi in the offshore and pelagic seawater and enriches the resource of marine fungi.