Abstract:Biological nitrogen fixation is an important N source to ecosystems, and nitrogen-fixing microorganisms play important roles in plant growth and development. Compared with symbiotic nitrogen-fixing microorganisms, asymbiotic nitrogen-fixing microorganisms are more widely distributed and more diverse, and they are of significance to the nitrogen cycle in the global ecosystem. In this review, the classification, phylogeny and community assembly of asymbiotic nitrogen-fixing bacteria were summarized. We compared the differences of asymbiotic nitrogen-fixing bacterial compositions and their nitrogen-fixing potential in different ecosystems (such as grassland, forest, ocean, farmland, etc.) and different parts of plants (such as canopy, phyllosphere, rhizosphere, endosphere, litter, etc.). The main factors affecting the community structure and nitrogen-fixing potential of asymbiotic nitrogen-fixing bacteria include climatic factors, soil properties and artificial measures. The frequently-used methods for studying asymbiotic nitrogen-fixing bacteria and their nitrogen-fixing potential were also summarized.

Abstract:Viruses play a key role in the biogeochemical cycle of global ocean by influencing the nutrient cycling, biodiversity, and genetic information transfer of microorganisms. Virus-encoded auxiliary metabolic genes (AMGs) can modulate the community composition and key metabolic pathways of microorganisms. Through the expression of AMGs, viruses reprogram the host metabolic pathways. Virus-encoded AMGs are known to include the genes associated with central carbon metabolism, nitrogen, phosphorus, and sulfur cycling, nucleotide metabolism, and oxidative stress response. AMGs are beneficial to the assembly and release of progeny virus, which is of great significance for virus reproduction and the research on virus-host interaction mechanism. In this paper, we briefly reviewed the origin, classification, and ecological roles of virus-encoded AMGs, aiming to provide reference for illuminating the roles of viruses in different ecosystems.

Abstract:Symbiotic bacteria can protect their insect hosts against pathogens by producing antimicrobial substances, regulating host immune-related genes, and competing with exotic pathogens for resources. To maintain the symbiotic relationship, insects have evolved fine-tuned regulatory mechanisms to avoid overactive immune responses to symbiotic bacteria, and the latter reduce or evade the damage from the host immune system via immune recognition signal polymorphisms or chemical mimicry. In this paper, we analyzed the function of symbiotic bacteria on host immunity and the mechanism and then explored the precise regulation of immune response by the host and the coevolution of the symbiotic system, hoping to provide a reference for further research on the influence of symbiotic bacteria on host immunity.

Abstract:Oncolytic virus (OV) therapy is an important anticancer means. Newcastle disease virus (NDV) displays effective oncolytic activity in many tumor types, which kills tumor cells in a selective way without being toxic to normal cells. In this paper, we reviewed the anti-tumor mechanisms of NDV from the aspects of inducing apoptosis and autophagy of tumor cells, inhibiting cell metabolism, stimulating immune response, and triggering ribosomal stress response in tumor cells, particularly the specific mechanism of inducing ribosomal stress response to regulate the translation system of tumor cells and trigger cell apoptosis. This review is expected to lay a solid theoretical foundation for in-depth research on the anti-tumor effect of NDV and targeted therapy of cancer in the future.

Abstract:Zinc cluster proteins, or Zn₂Cys₆ binuclear cluster proteins, are a family of transcription factors unique to the fungal kingdom. They are involved in diverse cellular processes of fungi, such as primary and secondary metabolism, stress response, and cell division. Zinc cluster proteins contain the DNA binding domain at the N-terminus, the regulatory domain in the middle, and the acidic activating domain at the C-terminus. The DNA binding domain, comprising a zinc finger motif, is responsible for binding to promoters of the target genes. At the moment, the three-dimensional structures of the DNA binding domains of some zinc cluster transcription factors have been resolved, and some proteins in this family have been verified to regulate the expression of multiple genes. However, no comprehensive report on the structures, dynamics and functions of them is available. Therefore, we analyzed the structural characteristics of different zinc cluster proteins, summarized the relationship between the domains and functions, and pointed out the future research focuses, hoping to provide a reference for in-depth research on zinc cluster proteins in the future.

Abstract:Vibrio parahaemolyticus is a food-borne pathogen that poses threats to food safety and human health around the world. It infects the host by directly injecting its effector proteins and mediates the exertion of virulence, and has evolved a perfect immune evasion strategy, causing diseases such as acute gastroenteritis, sepsis and necrotizing fasciitis. To promote the intracellular survival, V. parahaemolyticus invades epithelial cells and acidifies intracellular vesicles, and then it escapes into the cytoplasm before fusing with lysosomes and limits the production of reactive oxygen species. V. parahaemolyticus can induce autophagy and block caspase-1 activation mediated by the NLRC4 inflammasome. Besides, it can prevent the activation of MAPK and NF-κB signaling pathways by inhibiting TAK1 kinase to interfere with the immune system and achieve immune escape using a variety of ways. This article systematically summarized the immune evasion strategy of V. parahaemolyticus, which was critical for understanding its pathogenic mechanism, and provided new insights and directions for its possible immune evasion mechanism.

Abstract:Halides are important raw materials in agriculture, pharmaceutical industry, and chemical industry. They are mainly synthesized with chemical methods which, however, cause environmental pollution and have strick requirement for the reaction conditions. The key enzymes for the biosynthesis of natural halides are halogenases, including haloperoxidase (HPO), α-ketoglutarate dependent halogenase (KG-Hal), S-adenosylmethionine-dependent halogenase, and flavin-dependent halogenase (FDH). Among them, FDHs are the most promising halogenases owing to their wide distribution, substrate specificity, and halogenated regioselectivity. Therefore, we reviewed the structures, types, catalytic mechanisms, and applications of FDHs and summarized the use of gene analysis and gene screening to search for new FDHs. This review is expected to expand the knowledge of the types and catalytic mechanisms of FDHs, boost the research on FDHs and related enzyme engineering, and provide technical ideas for the synthesis of new halides.

Abstract:Metagenomics technology can directly extract all the microbial genetic material from environmental samples, without pure culture on the medium like traditional methods, which allows for in-depth understanding of the structures and functions of microbial communities. Moreover, it is of great significance to the diagnosis and treatment of diseases, management of the environment and understanding of life. All the genetic material of microorganism extracted from the environment is sequenced to obtain their reads which can be further assembled into contigs through the read assembly tools. Through binning of the contigs, more complete genes can be reconstructed from metagenomic samples. The effect of binning directly affects the subsequent biological analysis. Therefore, how to effectively bin these contigs containing different microbial genes has become a research hotspot and challenge in metagenomics. Machine learning methods are widely used in the binning of metagenomic contigs, which are generally classified into unsupervised contig clustering methods and supervised contig classification methods. This review introduced the methods for binning metagenomic contigs and analyzed the problems in binning methods such as low classification accuracy, high time cost, and difficulty in reconstructing more microbial genes from complex metagenomic datasets. Moreover, we summarized the future research on and development of the binning methods for metagenomic contigs. The authors suggested that semi-supervised learning, ensemble learning and deep learning methods should be used and combined with more effective data feature representation to improve the binning effect.

Abstract:Myeloid-derived suppressor cells (MDSCs) are heterogeneous immunomodulatory cells. In the instance of cancers, they exert immunosuppressive functions. To be specific, they induce T cell exhaustion and apoptosis through multiple pathways to promote the escape of tumor cells from destruction by the immune system and sustain cancer progression. Thus, they are the main obstacle in the fight against cancers. At the moment, MDSCs are the focus in development of anti-cancer drugs and the key targets of the drugs. It has been reported that polysaccharides reduced the count and proportion of MDSCs in cancer patients and tumor-bearing mice and eliminate the immunosuppression. Natural edible and medicinal fungal polysaccharides can activate immune response to tumors through multiple pathways, and there has been an explosion of research on the suppression of the immunosuppressive function of MDSCs. The currently available studies mainly focus on polysaccharides from Lentinula edodes and Ganoderma lucidum. In this review, we briefed the immunosuppressive mechanism of MDSCs in the cases of cancers, and then summarized the effects of edible and medicinal fungal polysaccharides on MDSCs, hoping to provide a new mindset for the development of anti-tumor drugs and auxiliary enhancement of immunotherapy such as immune checkpoint inhibitors with edible and medicinal fungal polysaccharides.

Abstract:[Objective] Ascosphaera apis is a lethal fungal pathogen of honey bee larvae. This study aims to identify and analyze alternative splicing (AS) and alternative polyadenylation (APA) of genes and long non-coding RNAs (lncRNAs) in A. apis spore (AaS) by PacBio single molecular real-time (SMRT) sequencing, further unveiling the complexity of AaS's transcriptome. [Methods] AS events of genes in AaS were identified with Suppa, and AS events of various types were confirmed based on RT-PCR. TAPIS pipeline was utilized to explore APA sites of AaS genes. MEME was employed to investigate statistics of sequences at 50 bp upstream of poly(A) splicing sites and identify the motifs. The lncRNAs were predicted based on CPC, CNCI, and Swiss-prot database, and the intersection was regarded as lncRNA dataset. Then the transcript length, exon number and length, intron length, GC content, and AS event number were compared between lncRNAs and mRNAs. [Results] A total of 2 609 AS events were identified in AaS, including 1 227 (47.03%) retained introns (RI), 842 (32.27%) alternative 3ʹ splice sites (A3), 415(15.91%) alternative 5ʹ splice sites (A5), 85 (3.26%) alternative first exons (AF), 35 (1.34%) skipping exons (SE), 4 (0.15%) alternative last exons (AL), and 1 (0.04%) mutually exclusive exon (MX). The reliability of various AS events such as RI, A5, SE, and A3 was validated via RT-PCR. Additionally, 5 552 genes containing APA sites were identified, among which genes with more than 5 APA sites were the most abundant (2 197), followed by genes with 1 APA site (1 149). The numbers of genes with 2, 3, 4, and 5 APA sites were 782, 596, 477, and 351, respectively. Moreover, upstream and downstream sequences of full-length transcripts in AaS had apparent base bias, with U and A respectively enriching at upstream and downstream. In total, 953 lncRNAs were identified, including 247 bidirectional lncRNAs, 171 long intergenic RNAs, 154 anti-sense lncRNAs, 141 sense lncRNAs and 9 intronic lncRNAs. Compared with mRNAs, these lncRNAs had fewer and shorter exons, shorter introns, shorter transcripts, lower GC content and fewer AS events. [Conclusion] In this study, we analyzed the AS, APA, and lncRNA in AaS. The findings enrich the basic biological information of A. apis, unravel the complexity of transcriptome in AaS, and lay a foundation for further exploration of the molecular function of various splicing isoforms in pathogenic spores and during pathogen infection.

Abstract:[Objective] Photobacterium damselae subsp. damselae (PDD) is a major pathogen which causes serious diseases in marine organisms. This study focused on two highly pathogenic PDD strains (PDD1605 and PDD1608) with strong hemolysis and phospholipase activities, which were isolated from maricultured fish in China. We analyzed the virulence and cytotoxicity of the extracellular products (ECPs) derived from the two PDD strains and their correlations with the strain pathogenicity. [Methods] The hemolysis and phospholipase activities of ECPs from PDD1605 and PDD1608 were measured on agar plates. The pathogenicity of the two live strains and their ECPs to Sebastes schlegeli were detected by artificial challenge test. Meanwhile, we prepared histopathological sections to observe the pathological damage of organs and tissues. Furthermore, we added the ECPs to the media of human embryonic kidney cell line HEK293T and mouse cardiac fibroblasts (MCFS) to study the toxicity of ECPs to mammalian cells.[Results] The live PDD1605 and PDD1608 strains showed strong pathogenicity to S. schlegeli. All the fish exposed to the strains at the concentration of 5×10⁸ CFU/mL died within 24 h. The ECPs of PDD1605 and PDD1608 also showed strong pathogenicity to S. schlegeli. The death curve trend was similar between ECPs and live strains. The ECPs caused serious tissue damage to S. schlegeli, which mainly included disintegration of intestinal mucosa, blood cell and inflammatory cell infiltration in liver, hemosiderosis in spleen, and inflammatory cell infiltration in myocardium. Additionally, the ECPs of the two PDD strains had strong hemolysis and phospholipase activities in vitro, which was basically consistent with the virulence characteristics of live strains. Moreover, they had cytotoxicity to HEK293T and MCFS cells, reducing the cell volume in vitro and causing degenerative changes such as round cell clusters.[Conclusion] TheECPs and live strains of PDD1605 and PDD1608 have highly consistent virulence and cytotoxicity. Our findings support that ECPs are the key pathogenic factor of highly pathogenic PDD strains.

Abstract:[Objective] This study aims to analyze the fungal community structure and functional groups under different moisture conditions in Bitahai Wetland, which is expected to serve as a reference for wetland resource management and ecological restoration. [Methods] Perennially submerged swamp wetland (SW), seasonally submerged swamp meadow (SM), and non-submerged meadow (M) in Bitahai Wetland, Southwest China were investigated. Based on Illumina high-throughput sequencing and FUNGuild, the soil fungal community structure and functional groups of the three types were analyzed and compared, and the influence of key environmental factors on the fungal community was probed. [Results] The alpha diversity of soil fungi showed no significant difference among SW, SM, and M. Non-metric multidimensional scaling and similarity analysis suggested that beta diversity was significantly different among the moisture gradients (R=0.501, P=0.001). Bitahai Wetland was dominated by Ascomycota, Basidiomycota, Rozellomycota, and Mortierellomycota. The abundance of Basidiomycota, Rozellomycota, and Mortierellomycota varied significantly across different moisture gradients (P<0.05). Pyronemataceae, Mortierellaceae, Archaeorhizomycetaceae, and Clavariaceae were the dominant soil fungal families (P<0.05). Correlation analysis indicated that soil pH, total nitrogen, nitrate nitrogen, ammonium nitrogen, iron, potassium, and sucrose, and plant PCoA1 were in positive correlation with alpha diversity of soil fungi (P<0.05). Redundancy analysis and heat map analysis showed that soil water content, ammonium nitrogen, urease, and plant Shannon index were the main causes of beta diversity variation (P<0.05). Variance partitioning showed that the fungal community was affected by both single environmental factors and the interaction among various environmental factors, especially the interaction of soil factors and plant community. The trophic types of soil fungi in the Bitahai Wetland were mainly saprotroph and saprotroph-symbiotroph. SW was dominated by endophytic-phytopathogenic fungi, and SM and M by undefined saprophytes. As the moisture decreased, pathotroph-saprotroph-symbiotroph increased and functional groups showed higher complexity.[Conclusion]Moisture influences the structure and functional groups of soil fungi in the Bitahai Wetland, and its soil fungal diversity and composition are influenced by multiple environmental factors. The influence of environmental factors on fungal diversity and fungal phyla is different.

Abstract:[Objective] This study investigated the temporal and spatial changes of bacterioplankton community in Zhejiang and Fujian coastal ecological pasture technology demonstration areas through four voyages in different seasons, aiming to explore their potential impact on the marine ecosystem. [Methods] The 16S rRNA gene V4 region of bacterioplankton was sequenced for 128 samples from four seasonal voyages in two demonstration areas. The composition, diversity and functions of bacterioplankton were compared among different seasons and between different demonstration areas.[Results] A total of 2 510 976 high-quality bacterioplankton sequences were obtained from 128 samples after quantization. The main dominant bacterioplankton groups in the two demonstration areas were Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, Actinobacteria, Flavobacteria, etc. The α-diversity index of the bacterioplankton in the two demonstration areas fluctuated with seasonal succession and had significant correlations with most of the environmental factors. The bacterioplankton community assembly in Zhejiang and Fujian coastal areas varied over time, and diffusion restriction was the most important ecological process in the two demonstration areas (except the Taishan island demonstration area in autumn). This finding was confirmed by the distance decay of Bray-Curtis similarity in the two demonstration areas. The prediction with functional annotation of prokaryotic taxa (FAPROTAX) demonstrated that the bacterioplankton in the two demonstration areas were mainly involved in chemoheterotrophy and nitrogen/sulfur cycle, which were associated with the changes of local environmental factors to a certain extent. [Conclusion]The structure of the bacteiroplankton community in Zhejiang and Fujian coastal ecological pasture technology demonstration areas was affected by geographical distance, physicochemical factors, and seasons. The potential ecological functions of bacterioplankton dominated by chemoheterotrophs varied among different seasons.Revealing the changes of potential ecological functions and community assembly of bacterioplankton provides theoretical support for the future development of marine pasture at the microbial level.

Abstract:[Objective] To analyze the serotypes, antibiotic resistance, and resistance genes of 21 isolates of Glaesserella parasuis in large-scale pig farms.[Methods] Serotypes were identified by PCR and the resistance to 25 antibiotics with the K-B disk diffusion method. Seven resistance genes, including bla-_TEM, bla-_NDM, and bla-_CTX, were detected by PCR. Then chi-square test and Fisher's exact test were used to analyze the association between resistance phenotypes and genotypes. The β-lactam resistance gene bla-_TEM was sequenced and the sequences of amino acids encoded by bla-_TEM from different isolates were compared. Finally, the relationship between the differential sites of the amino acids and antibiotic resistance was analyzed by CLC Sequence Viewer. [Results] The isolates were dominated by serotypes 4 and 12. They showed high rate (61.9%, 13/21) of resistance to β-lactam antibiotic oxacillin and 90.5% (19/21) of the isolates demonstrated multi-antibiotic resistance. bla-_TEMwas identified in 52.4% (11/21) of the isolates, which was significantly correlated with the resistance phenotype of penicillin G, oxacillin, and cefradine. There were mutations of the amino acids encoded by bla-_TEM among isolates, which may be related to the antibiotic resistance of the isolates.[Conclusion]Multi-antibiotic resistance of G. parasuis isolates is prevalent in large-scale pig farms, and the high rate of β-lactam resistance is attributed to the inborn resistance gene bla-_TEM. The conclusion lays a theoretical basis for surveillance of antibiotic resistance of G. parasuis in large-scale pig farms.

Abstract:[Objective] Sulphide (H₂S) in mariculture seriously damages the health of cultured organisms. Controlling the metabolic activity of sulfate-reducing bacteria (SRB) in mariculture is an effective way to inhibit H₂S production. [Methods] In this study, SRB strains were enriched and isolated from the mariculture sediments via dilution coating-repeat dish sandwish cultivation method. The H₂S activity was inhibited by adding nitrate. [Results] Two SRB strains Desulfovibrio sp. NY-1 and Clostridium sp. NH-1 were isolated, which respectively accumulated up to 435 and 150 mg/L H₂S at 35℃, pH 7.0, and salinity of 20-30 mg/L. The H₂S activity of NY-1 was not effectively inhibited by nitrate, which was related to the gene regulation and the lack of the enzyme system using nitrate as the electron acceptor. Nitrate reversibly inhibited the H₂S activity of NH-1 which had the ability of dissimilatory nitrate reduction to ammonium (DNRA) and preferentially used nitrate as the electron acceptor. Nitrite, the intermediate metabolite of DNRA, was the main factor for inhibiting the H₂S production activity of NH-1 since it can inhibit the growth and reproduction of the strain. [Conclusion] The inhibition mechanisms and effects of nitrate vary among different SRB strains. Before controlling sulfide pollution, we need to analyze and distinguish the sulfide-producing bacteria.

Abstract:[Objective] Probiotics producing β-mannanase were isolated from the intestine of Cyprinus carpio. [Methods] The strain with high β-mannanase production was screened based on the transparent hydrolysis circle on the screening plate and shake flask fermentation. Through morphological observation, physiological and biochemical tests, 16S rRNA gene sequencing, and comparative genomics-based analysis, the strain was identified. The enzyme activity was evaluated with the dinitrosalicylic acid (DNS) method. The tolerance to temperature, acid, and bile salt, and bacteriostasis (Oxford cup method) of the strain were tested, and then the biosafety was evaluated with the filter paper method and intraperitoneal injection method. [Results] A total of 62 strains producing β-mannanase were isolated from C. carpio, among which HF-14109 was prominent in the enzyme production and was identified as Bacillus velezensis. The optimal reaction temperature and pH for the enzyme were 45 and 6.0. It was stable at 20-80 and pH 4.0-9.0. Cu²⁺, Fe³⁺, Zn²⁺, and Ba²⁺ can activate the enzyme, while Mn²⁺ and Ca²⁺ inhibited the enzyme. It can degrade konjac powder, guar gum, and sophora bean gum. HF-14109 showed tolerance to acid, high temperature, and bile salt and suppressed Escherichia coli EC 05, Edwardsiella tarda ET 03, Staphylococcus aureus SA 16, Aeromonas hydrophila Ah 01, among other bacteria. No death or abnormality occurred to the C. carpio injected with 10⁷-10⁹ CFU/mL HF-14109 suspension. It demonstrated no resistance to gentamicin, kanamycin, clindamycin, chloramphenicol, erythromycin, streptomycin, tetracycline, vancomycin, and ampicillin. [Conclusion] HF-14109 with high production of β-mannanase was isolated from C. carpio and the enzyme demonstrated high activity, good enzymatic properties and probiotic properties, and safety. HF-14109 can be used to remove mannan in aquatic feed.

Abstract:[Objective] Aminopeptidase gene pepN from Bacillus licheniformis E7 was cloned into Escherichia coli BL21 for heterologous expression of recombinant aminopeptidase EcPepN. The enzymatic properties of the recombinant EcPepN were studied. The combination of EcPepN with alkaline protease efficiently improved the hydrolysis of soybean protein and casein and released multitudinous peptides and amino acids. [Methods] With the genomic DNA of E7 as template, the aminopeptidase gene pepN was cloned into the expression vector pET28a to construct recombinant expression plasmid pET28-pepN, which was transformed into the competent cells of E. coli BL21. Through DNA sequencing verification, the recombinant E. coli BL21/pET28-pepN was obtained. The recombinant enzyme was purified by nickel-affinity chromatography. The pH and temperature stability, half-life, and NaCl tolerance of the pure enzyme were tested. With the commercial aminopeptidase combined with alkaline protease as a control, the combination of EcPepN and alkaline protease was used to hydrolyze soybean protein and casein, and the constituents of active small peptides and free amino acids in the hydrolysate were determined. [Results] The recombinant EcPepN was expressed in E. coli BL21. SDS-PAGE analysis showed that the purified target protein presented a single band at about 52 kDa. Among the seven substrates tested, the preferred substrate of EcPepN was Ala-pNA. Under optimal conditions (pH 9.0 and 50. After 22 days of incubation in 3.0 mol/L NaCl, the residual activity was more than 55%. When the recombinant aminopeptidase EcPepN worked synergistically with alkaline protease to hydrolyze soybean protein and casein, its hydrolysis efficiency was similar to that of the combination of commercial aminopeptidase and alkaline protease. More than 70% of the constituents of hydrolysate were peptides with molecular weight of less than 500 Da and it also contained more than 2 000 mg/L of free amino acids. [Conclusion] The recombinant strain containing aminopeptidase EcPepN was developed. The recombinant EcPepN shows pH and temperature stability and strong tolerance to NaCl of high concentration. It can efficiently catalyze the hydrolysis of soybean protein and casein and release small molecular active peptides and abundant free amino acids. This study provides important methods for enhancing deep protein hydrolysis and improving the values of protein-rich bioresources.

Abstract:[Objective] To explore the diversity of arbuscular mycorrhizal fungi (AMF) in root zone of Amorphophallus konjac with and without soft rot under different durations of continuous cropping.[Methods] A specific primer pair (AMV4.5NF/AMDGR) targeting the 18S small subunit rRNA gene of AMF was used for PCR amplification of the DNA from the root and rhizosphere soil samples of diseased and healthy A. konjac plants under non-continuous cropping and 2 and 3 years of continuous cropping, respectively, and the DNA library was constructed. Based on high-throughput sequencing and bioinformatics analysis, the relationship between soft rot and AMF diversity in the root zone of A. konjac was investigated. [Results] AMF infection occurred in the roots of A. konjac with specific structures such as hyphae, vesicles, and arbuscules. The total infection rate, infection intensity, and spore density of AMF in healthy plants were significantly higher than those in diseased plants under the same continuous cropping duration (P<0.05). Both the total infection rate and infection intensity of AMF in diseased plants decreased remarkably with the prolongation of continuous cropping. A total of 53 species in 9 genera of AMF (49 known species and 4 novel species) were identified from all samples. Glomus and Paraglomus were the dominant genera of AMF, accounting for 41.5% and 26.4% of the total AMF community, respectively. Among the species identified, Paraglomus sp. VTX00308 showed the highest relative abundance (12.3%) and was shared by all samples. Continuous cropping duration, soft rot, and their interaction significantly affected the Shannon and Simpson indices of AMF in the roots and the Chao1 of AMF in the rhizosphere soil (P<0.05). Six AMF species considerably varied in relative abundance after soft rot occurred under continuous cropping (P<0.05). Difference in AMF species composition, relative abundance, and community structure was revealed between diseased and healthy plants under different continuous cropping durations by non-metric multidimensional scaling. Correlation analysis indicated that the incidence and severity index of soft rot were negatively correlated with the Shannon index (root and rhizosphere soil), Chao1 (root), Simpson index (root), total infection rate, infection intensity, and spore density of AMF in the root zone of A. konjac (P<0.01). [Conclusion] AMF spore density in the rhizosphere soil and AMF infection rate, species number, and community diversity in the roots of A. konjac plants with soft rot all decreased compared with those of the healthy plants, leading to remarkable shifts in the root-zone AMF community structure under continuous cropping. Our results suggest that soft rot occurrence during continuous cropping may reshape the AMF community in the root zone of A. konjac through altering the species composition, relative abundance, and diversity of AMF in both the roots and rhizosphere soil.

Abstract:[Objective] Increasing studies have proved that probiotics can regulate the central nervous system through the microbiome-gut-brain axis and affect the occurrence and development of mental disorders. Therefore, the development of probiotics resources related to mental health is of great significance. We employed the mouse model of chronic unpredictable mild stress (CUMS) to study the alleviating effects of Lactobacillus kefiranofaciens 1207 isolated from Tibet Kefir grains on the anxiety and depression behavior in mice. [Methods] The CUMS model was constructed and intragastrically administrated with L. kefiranofaciens 1207 (10⁹ CFU/d) for two weeks. The effects of the strain on the anxiety-like and depression-like behaviors of mice were evaluated by open field test, tail suspension test, and forced swimming test. The regulatory effects of the strain on tryptophan metabolism, hypothalamus-pituitary-adrenal axis, and inflammatory cytokines were evaluated based on the content of biomarkers in hypothalamus, serum, and spleen. [Results] Compared with the control, L. kefiranofaciens 1207 prolonged the retention time in the central region in the open field test (P<0.05), as well as the stationary time in the tail suspension test (P<0.01) and forced swimming test (P<0.05). Additionally, it improved the tryptophan metabolism of hypothalamus by reducing 5-hydroxyindoleacetic acid/5-hydroxytryptamine in mice and regulated the balance of hypothalamus- pituitary-adrenal axis by lowering the serum level of corticosterone. By up-regulating the relative expression of anti-inflammatory cytokine (inflammatory cytokines-10, IL-10) in mouse spleen (P<0.05), L. kefiranofaciens 1207 inhibited the occurrence of inflammation. [Conclusion] Two weeks of oral administration of L. kefiranofaciens 1207 can effectively regulate the biomarkers related to the gut-brain axis and alleviate the anxiety and depression-like behavior of mice. Our study provides new probiotic intervention strategies for the prevention and treatment of psychiatric disorders.

Abstract:[Objective] The ubiquitous air-liquid interfaces affect bacterial motility and nutrient transport, thereby regulating the interaction among microbial populations and microbial community structure. Therefore, it is of vital importance for understanding and elucidating the mechanisms of microbial diversity generation and maintenance as well as the ecological functions to clarify the movement characteristics of microorganisms at microscopic interfaces. [Methods] With microfluidic microscopes (ultra-high speed fluorescence microscope and digital holographic microscope), we quantified the movement patterns of Pseudomonas aeruginosa (PAO1) cells near air-liquid-solid and air-liquid interfaces of droplet. [Results] Below the air-liquid interfaces, the trajectories of PAO1 are as follows:straight lines, clockwise circles, or counterclockwise circles with R_min(minimum radius of curvature)=3 µm. At the air-liquid-solid interfaces, 6.45% of the immobile cells accumulated at the edge of the interfaces and completed irreversible attachment directly. Meanwhile, due to capillary flow and Marangoni effect inside the droplet, mobile cells returned in the direction perpendicular to the interface or moved in the direction approximately parallel to the interface and attached after swimming in a straight line to a region within about 40 µm from the interface. These behaviors significantly modulated the spatial distribution of PAO1, promoting the migration toward the air-liquid-solid interface. Therefore, the active flagellar motility played a little role in the process. [Conclusion] With similar trajectories in both the solid-liquid interfaces and the air-liquid interfaces, PAO1 can move towards and subsequently attach onto the air-liquid-solid interfaces under the complex cell-surface interfacial forces.

Abstract:[Objective] Citrulline is a precursor of ethyl carbamate in fermentation systems. To reduce the concentration of citrulline, we established a putative transport protein (PTP)-based high-throughput screening strategy to isolate and identify citrulline-utilizing strains from different samples and then evaluated the extracellular citrulline utilization abilities of the strains. [Methods] We compared the amino acid sequences of PTPs to determine the motif and then designed degeneracy primers to obtain the nucleic acid sequences of PTPs. The citrulline-utilizing strains were isolated by high-throughput screening strategy combined with bromocresol purple chromogenic circle method, colony PCR with degeneracy primers, and diacetyl monoxime colorimetric method. [Results] A pair of degeneracy primers was designed for the screening of citrulline-utilizing strains. Using the high-throughput screening strategy, we isolated 65 citrulline-utilizing strains from different environments. Levilactobacillus brevis PC4 consumed 91.08% citrulline in 4 h. The PTP genes of four strains were located in arc gene cluster, which indicated that the function of PTP was closely related to citrulline metabolic pathway. [Conclusion] PTP is an important functional protein for extracellular citrulline utilization of bacteria. High-throughput screening strategy based on PTP gene is an efficient way to isolate citrulline-utilizing strains from different environments.

Abstract:[Objective] To identify the key host protein that can regulate the replication of porcine epidemic diarrhea virus (PEDV). [Methods] We usedliquid chromatography with tandem mass spectrometry (LC-MS/MS) technology combined with tandem mass tag (TMT) to analyze the proteomic differences between PEDV-infected Vero cells and the uninfected group at 36 h post inoculation. A total of 114 significantly differentially expressed proteins were identified, of which 5-hydroxymethylcytosine binding, ES cell-specific protein (HMCES) was significantly up-regulated. The eukaryotic expression plasmid of HMCES was further constructed, and the effect of HMCES overexpression on PEDV replication was examined by Western blotting and real-time fluorescent quantitative PCR. The specific siRNA against HMCES gene was synthesized, and Western blotting and RT-qPCR were employed to detect the effect of siRNA on HMCES expression and the effect of interfered HMCES on PEDV replication. [Results] Overexpression of HMCES significantly promoted PEDV replication in Vero cells, and the level of replication increased in a dose-dependent manner. siRNA-341 down-regulated the expression of endogenous HMCES to inhibit PEDV replication. [Conclusion] HMCES promotes PEDV replication in Vero cells. This study provides a reference for further exploring the role and mechanism of HMCES in the anti-PEDV immune response.

Abstract:[Objective] To investigate the action mechanism of scorpion venom peptide Ctry2459 against Candida albicans. [Methods] The broth microdilution and plate count methods were used to determine the minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of Ctry2459 against C. albicans. Time-kill curves were drawn based on the plate count method. In addition, we evaluated the influence of Ctry2459 on the integrity of the cell membrane of C. albicans by propidium iodide (PI) absorption experiment. DNA gel retardation assay was conducted to investigate the binding effect between Ctry2459 and nucleic acid. Also, we measured the effects of Ctry2459 on the reactive oxygen species (ROS) level, mitochondrial membrane potential, and apoptosis/necrosis induction of C. albicans by flow cytometry assays. [Results] The MIC and MFC of Ctry2459 against C. albicans were 25 μg/mL and 50 μg/mL, respectively. Ctry2459 killed C. albicans in a time- and concentration-dependent manner and mainly through membrane disruption, and it could also induce C. albicans necrosis via a ROS-dependent pathway. [Conclusion] Ctry2459 had the potential for the development of anti-C. albicans drugs.

Abstract:[Objective] To clarify the mechanism of Ubr1 affecting the polar growth of Beauveria bassiana based on the differentially expressed genes (DEGs) and proteins (DEPs) and their pathways in the conidia of the wild-type (WT) and ubr1-deleted (Δubr1) strains at the same time point and thus to lay a theoretical basis for improving potential of B. bassiana for biocontrol. [Methods] Through KEGG analysis of the transcriptome and proteome, the pathways of the DEGs and DEPs were clarified. The germinated conidia in each germination medium (GM)-derived plate were photographed with a microscope to verify the influence of the differential pathways on polar growth of the conidia.[Results] ubr1 deletion impaired the germination of conidia, resulting in abnormally curved or hook-shaped germ tubes. Both the DEGs and DEPs were involved in nitrogen metabolism, arginine and proline metabolism, and ether ester metabolism. Further verification showed that abnormal arginine metabolism caused by ubr1 deletion was an important reason for the disorder of conidial polar growth and that abnormal metabolism of galactose and nitrogen led to slow germination of conidia. [Conclusion]The absence of ubr1 blocks arginine metabolism, resulting in abnormal polar growth of germ tube. The abnormal metabolism of galactose and nitrogen also delays the germination of conidia. The findings are expected to enhance the understanding of the mechanism of polar growth and the penetration process in the infection cycle of filamentous fungi.

Abstract:Pulcherriminic acid synthesized and secreted by Bacillus licheniformis is an iron chelator that helps to maintain iron homeostasis by removing Fe³⁺ from the environment. [Objective] This study is to investigate the regulatory mechanisms of DegU in the synthesis and secretion of pulcherriminic acid. [Methods] Based on the native strain B. licheniformis DW2 and its derivative strains DW2ΔdegU (degU deletion) and DW2::Pbay-degU (degU overexpression), this study excavated the regulatory mechanisms of DegU in the synthesis and secretion of pulcherriminic acid by determining pulcherriminic acid content, real-time quantitative polymerase chain reaction (RT-qPCR), electrophoretic mobility shift assay (EMSA), and green fluorescent protein (GFP) expression assay. [Results] The pulcherriminic acid yield of DW2ΔdegU was 56.8% higher than that of DW2, while the yield of DW2::Pbay-degU was 83.7% lower than that of DW2. In addition, after degU deletion, compared with the condition of DW2, the transcriptional levels of pulcherriminic acid synthetase genes yvmC and transporter gene yvmA increased to 2.85 and 2.71 times, respectively, whereas the transcriptional level of yvmB, another negative regulator gene of yvmC and yvmA, decreased to 0.35 times. In DW2::Pbay-degU, the transcription levels of yvmC and yvmA reduced to 0.47 and 0.24 times, respectively, while the transcription level of yvmB rose to 1.78 times that of DW2. EMSA and GFP expression assay showed that DegU could directly bind to PyvmC and PyvmB promoters, but had no direct interaction with the promoter of yvmA (PyvmA). [Conclusion] DegU regulated the synthesis and secretion of pulcherriminic acid in two ways:on the one hand, DegU negatively regulated yvmC by directly binding to the promoter of yvmC-cypX cluster; on the other hand, DegU activated the expression of yvmB and negatively regulated yvmC-cypX and yvmA in an indirect way.

Abstract:[Objective] Acidithiobacillus ferrooxidans is one of the key bacteria involved in the bioleaching of primary and secondary ores and solid wastes. In the bioleaching environments, high levels of heavy metals are one of the major challenges encountered by A. ferrooxidans. Resistance to heavy metals stress is essential for the adaptation of microorganisms to the extreme environments. However, the tolerance and iron oxidation response of A. ferrooxidans to Ni²⁺ stress remain unclear. [Methods] In this study, we employed a two-step culture system with the Ni²⁺concentration range designed within 0-40 g/L. The ferrous oxidation efficiency, expression of rus operon genes, and extracellular electron transfer of A. ferrooxidans exposed to Ni²⁺ stress were respectively investigated by phenanthroline spectrophotometry, real-time quantitative PCR, and cyclic voltammetry. [Results] A. ferrooxidans could tolerate Ni²⁺ stress at the concentration ≤30 g/L in the two-step culture system. However, 40 g/L Ni²⁺ significantly inhibited the ferrous iron oxidation activity of A. ferrooxidans, and the ferrous oxidation inhibition efficiency at the time point of 24 h was 59.4% that of the control (0 g/L Ni²⁺). Moreover, the high concentration (40 g/L) of Ni²⁺ decreased the transcriptional levels of rus operon genes. Especially, the transcriptional levels of cyc1 and coxBACD were maximally down-regulated by 16 folds and 2.7-7.4 folds, respectively. Meanwhile, the rate of extracellular electron transfer in A. ferrooxidans exposed to 40 g/L Ni²⁺ was decelerated to 24.5 µA in comparison to the control (0 g/L Ni²⁺, 29.0 µA). [Conclusion]High concentration (40 g/L) of Ni²⁺ down-regulated the expression of rus operon genes involved in Fe²⁺oxidation, inhibited the electron transport from Fe²⁺ oxidation, and finally slowed down ferrous oxidation activity. The findings provide theoretical support for the bioleaching of Ni-containing solid waste.

Abstract:[Objective] Sorbicillinoids are important natural active compounds synthesized by Trichoderma reesei and exhibit various biological activities, such as antineoplastic, antioxidative, and antiviral properties. In this study, we aimed to clarify the biological functions and regulatory mechanisms of TrSet2 during sorbicillinoid biosynthesis in T. reesei. [Methods] Weidentified the gene encoding TrSet2 in T. reesei using bioinformatic analysis, and investigated its functions in sorbicillinoid biosynthesis by knockouting and overexpressing Trset2 gene. In addition, the transcription activator Ypr1 was overexpressed in Trset2 knockout strain to elucidate the regulatory mechanism between TrSet2 and Ypr1. [Results] Knockout of Trset2 led to the strain's inability to synthesize sorbicillinoids. Conversely, overexpression of Trset2 resulted in a significant increase in sorbicillinoid secretion. Further studies showed that overexpression of Ypr1 in Trset2 knockout strain restored sorbicillinoid production. [Conclusion] TrSet2 positively regulated the sorbicillinoid biosynthesis via controlling the expression of Ypr1. Our results facilitated the control of sorbicillinoid secretion during the fermentation of T. reesei and the development of cell factories for high-level synthesis of sorbicillinoids based on the regulatory mechanism.

Abstract:[Objective] To activate the biosynthesis gene cluster for polyketide synthases-nonribosomal peptide synthetases (PKS-NRPS) from the marine-derived Arthrinium arundinis ZSDS1-F3 based on genome analysis, and to isolate and identify the secondary metabolites. [Methods] The cryptic biosynthesis gene cluster was activated by promoter engineering and heterologous expression. The products were isolated and elucidated by HR-ESI-MS and 1D and 2D NMR, and the biosynthetic pathways of them were deduced based on gene recombination and bioinformatics analysis. [Results] A dormant PKS-NRPS-encoding gene cluster from A. arundinis ZSDS1-F3 was activated through heterologous expression in Aspergillus nidulans A1145. Two new products were isolated and the biosynthetic pathways were clarified. [Conclusion] Via bioinformatics analysis, genome mining plays an important role in the discovery of novel natural products from fungi.

Abstract:[Objective] Streptococcus suis is a major swine pathogen and a zoonotic agent. Swine tonsil is a natural habitat of S. suis, and the tonsillar S. suis from healthy pigs is considered as an important source of infection for susceptible pigs and humans. Thus, the epidemiological investigation of S. suis from healthy pigs in slaughterhouses is of great significance for public health. [Methods] We collected healthy pigs' tonsils from a slaughterhouse in Zhejiang in 2020-2021, isolated and identified S. suis, and serotyped the strains by the serotype-specific PCR assay. Through the resistance gene detection, antimicrobial susceptibility testing, and zebrafish infection experiment, we examined the antimicrobial resistance and pathogenic characteristics of these isolates. [Results] The positive rate of S. suis in 131 tonsil samples of healthy pigs was 62.59% (82/131), and we isolated a total of 68 strains. The strains were dominated by serotype 16 (16.18%, 11/68), followed by serotype 31 (11.76%, 8/68), serotype 9 (7.35%, 5/68), and serotype 3 (7.35%, 5/68). In addition, tonsil samples containing 2 or more serotypes of S. suis accounted for 15.85% (13/82). The antimicrobial susceptibility testing showed that the isolates were mainly resistant to lincosamides (100%, 68/68), macrolides (98.53%, 67/68), and tetracyclines (100%, 68/68), and all isolates were multidrug-resistant. It is worth noting that 18 isolates were resistant to penicillin, 3 resistant to cefotaxime, 2 resistant to rifampicin, and 11 resistant to linezolid. The detection rate of both macrolides/lincosamides resistance genes and tetracycline resistance genes was 82.35% (56/68), which was the main reason why these isolates were resistant to the above antimicrobials. We selected 25 representative strains for the zebrafish infection experiment, and the results showed that 5 strains were highly virulent with mortality of 80%-100% when challenged with the dose of 3×10⁶ CFU/fish. [Conclusion] Healthy pigs in this area had a high detection rate of S. suis. All isolates were multidrug-resistant, and some were highly virulent. These results contribute to understanding the incidence of S. suis and formulating relevant prevention and control strategies in this area.

Abstract:[Objective] To investigate the effect of modified mouse cathelicidin related antimicrobial peptide (CRAMP) combined with antibiotics against the mature biofilm of Pseudomonas aeruginosa PAO1 and thereby lay a theoretical basis for the combined application of anti-biofilm drugs. [Methods] Broth microdilution was employed for the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) of CRAMP and antibiotics on PAO1. The time-kill curve (TKC) method was used to determine the bactericidal activities of CRAMP alone, antibiotics alone, and the combination of CRAMP and antibiotics against the mature biofilm of PAO1. Colony counting method and confocal laser scanning microscope (CLSM) were applied to evaluate the dispersal effect of CRAMP combined with antibiotics on the biofilm. [Results] Compared with the antibiotics alone, the combination of antibiotics (except for two carbapenems and four β-lactam antibiotics) with CRAMP recorded the decrease in MBEC, particularly vancomycin, roxithromycin, and azithromycin (1/4 that of antibiotics alone). According to the TKC, the combination of vancomycin, roxithromycin, and azithromycin with CRAMP showed faster and stronger bactericidal activity than antibiotics alone, especially the combination of vancomycin which killed all (100%) biofilm bacteria in 3 h. Under CLSM, the number, volume, area, and fluorescence intensity per unit area of biofilm were significantly changed. [Conclusion] CRAMP can disperse the mature biofilm of PAO1 and has synergistic effect with vancomycin, and the synergistic effect is the most significant at 1 h.