Abstract:Zearalenone (ZEN), one of the common mycotoxins in moldy grain crops such as maize and wheat, brings health hazards to domesticated animals and humans, demonstrating reproductive toxicity, immunotoxicity, hepatotoxicity, and nephrotoxicity. Physical, chemical, and biological methods have been used to address zearalenone contamination. The physical and chemical methods applied in feed production have a risk of secondary contamination. Biodegradation as a detoxification method using microbial adsorption and degradation of zearalenone is the characterized by high safety, environmental friendliness, high efficiency, specificity, and high detoxification rate and does not affect the nutritional value of grains. Therefore, biodegradation becomes a hot spot for the research on zearalenone degradation. This article mainly introduces the microbial species capable of degrading zearalenone from the detoxification capacity, methods, and products and summarizes the advantages and prospects of microbial detoxification, aiming to provide new perspectives for the theoretical research and practical application of microbial methods for degrading zearalenone.

Abstract:The co-pollution of heavy metals and organic compounds is one of the major environmental issues today. Understanding the degradation and transformation characteristics of chlorobenzenes (CBs) under the influence of heavy metals is of great significance for remediation of the co-pollution. We summarized the current research status of CB biodegradation, clarified the main functional bacterial genera including Burkholderia, Pseudomonas, Dehalobium, and Dehalococcoides involved in CB degradation, and then outlined the co-pollution status of heavy metals and CBs. The co-pollution of heavy metals and CBs exists in the vast majority of pollution cases. Further, we systematically reviewed the effects of typical heavy metals on the biotransformation and degradation of CBs. Most heavy metal ions exert inhibitory effects on the biotransformation and degradation of CBs under aerobic or anaerobic conditions, and their inhibitory effects are significantly influenced by pH and the species, concentration, and valence state of heavy metals. In addition, the mechanisms of CB transformation and degradation under the influence of heavy metals were analyzed. A molecular mechanism model was constructed with consideration to three influencing mechanisms. Finally, we analyzed the current problems and limitations and prospected the future development direction, aiming to provide support for the remediation of heavy metal-organic co-pollution.

Abstract:Malignant tumors are a major global public health problem threatening human health. The combination of multiple therapies, especially targeted therapy combined with immunotherapy, can slow down the progress of malignant tumors and improve the short-term survival rate of the patients. However, these treatment methods cannot cover all patients and have limited long-term efficacy. Therefore, how to improve the quality of life and long-term survival rate and reduce mortality of the patients has become a key problem to be solved. Increasing studies have demonstrated that the distribution of intestinal microorganisms is associated with the occurrence and development of malignant tumors, and regulating intestinal microbiota can serve as a new adjuvant therapy for malignant tumors. Despite the extensive studies about Akkermansia muciniphila in tumor treatment, little is known about the safety and effectiveness of A. muciniphila in the adjuvant treatment of malignant tumors. Therefore, we summarized the research results and application results of A. muciniphila by reviewing the studies on malignant tumors in recent years, aiming to enrich the choices for the comprehensive treatment in clinical practice.

Abstract:With the in-depth study of the relationship between intestinal microbiome and host and the rapid development of genetic engineering, the application of genetically engineered bacteria (GEB) in the medical field has become a research hotspot. GEB refer to the bacteria that have been genetically engineered to efficiently express exogenous proteins or compounds to achieve specific goals. Compared with traditional drugs, GEB have a variety of advantages. The construction process of GEB includes the selection of chassis, the acquisition of functional genes, and gene transfer and recombination. At present, the bacteria serving as GEB chassis can be classified into two categories:generally regarded as safe (GRAS) strains and commensal strains. The application of multi-omics facilitates the selection of chassis. Functional genes can be obtained by PCR, DNA synthesis, CRISPR-Cas9 or Red/ET recombination system according to their sizes. Heat shock and electroporation are widely used for plasmid transfer in bacteria. Homologous recombination can directly integrate the target gene into the host's chromosome. The encapsulation technology can improve GEB's survival rate and colonization ability, and synthetic gene circuits can make GEB intelligent. Functional stability, effectiveness, and safety are the general indicators for evaluating GEB. The instability of plasmid is an inherent defect of the GEB constructed by plasmid-mediated gene transfer, while the GEB constructed by gene integration have strong stability. In addition, to achieve long-term stable expression of functional genes, researchers need to evaluate and alleviate the impact of load. The efficacy and safety of GEB need to be evaluated in vitro, in animal models and clinical trials. There are some methods that have successfully achieved optimization of the above indicators. GEB have been widely used in the diagnosis and treatment of inflammatory diseases, tumors, metabolic diseases, infectious diseases, neurological diseases, and other diseases, playing a unique role. However, there are still problems regarding construction methods, performance evaluation and optimization, and large-scale production, which limit the clinical application. In this paper, we introduced the methods for construction and performance evaluation of GEB, summarized the application in disease diagnosis and treatment in recent years, pointed out the existing problems, and prospected the development of this field in the future.

Abstract:Germ-free animals refer to the animals in which bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, rickettsia, viruses, protozoa, and parasites cannot be detected in any part of the body and in vitro by modern technologies. Since germ-free animals do not carry any microorganism, they can be modeled into the animals carrying specific microorganisms. Because of the dormant immune system, germ-free animals are extremely sensitive to microbial infections. A variety of gnotobiotic animal models can be established for specific microbial infection experiments and pathogenic mechanism research. As a key tool, germ-free animals are pivotal in studying the relationship between microbiome and diseases and play an irreplaceable role in the research on the relationship between microbiome and host health and the mechnisms of infections. We briefly introduce germ-free animals and review the applications of germ-free animals in the research on the mechanisms of host-microorganism interactions.

Abstract:[Objective] A targeted mutation system was established in Pichia pastoris to provide an efficient editing tool for genetic engineering.[Methods] A cytosine base editor (CBE) was designed with nCas9 derived from the clustered regularly interspaced short palindromic repeats/Cas9 nuclease (CRISPR/Cas9) system and a cytosine deaminase PmCDA1. A cytosine-rich sequence in the yeast genome was selected as the target to validate the CBE activity. High-throughput sequencing was employed to analyze the editing efficiency and mode of the CBE. Furthermore, we explored how factors such as the length of linker peptides, the relative position of fusion proteins, and the length of gRNA targeting sequence affect the CBE function. [Results] The CBEs established by the fusion of nCas9 and PmCDA1 were capable of editing the cytosine in the P. pastoris genome. The highest editing efficiency was achieved with a linker peptide of (GGGGS)₁₀, and the editing window was located between C20 and C14 at the distal end of the protospacer adjacent motif. The window location at C18 achieved the maximum editing efficiency of 85.1%. Changes in the relative position of nCas9 and PmCDA1 had minor effects on the editing efficiency and modes of CBEs. However, the length of gRNA targeting sequence affected the editing efficiency of CBEs and cannot be shorter than 17 nt. The gRNA targeting sequences of 19-23 nt were capable of guiding CBEs to edit the genome. [Conclusion] This study establishes a set of CBEs with high editing efficiency in P. pastoris, which serves as a solid foundation for further genetic engineering.

Abstract:[Objective] To investigate the community characteristics, network complexity, and community stability of arbuscular mycorrhizal fungi (AMF) in the soil applied with reduced phosphorus (P) fertilizer combined with manure, and reveal the short-term effects of soil properties on the network characteristics and community stability of AMF community in the context of manure substitution for chemical fertilizers. [Methods] On the basis of the long-term inorganic P fertilizer application experiment started in 2012, a split plot experiment was designed with the application of reduced P fertilizer combined with manure and started in 2018. Six treatments were designed:inorganic P supply at 0, 75, and 150 kg/hm² and 30% reduction in inorganic P fertilizer (i.e., 0, 52.5, and 105 kg/hm²) combined with manure at 3 187 kg/hm², with three replicates for each treatment. High-throughput sequencing and bioinformatics analysis were employed to explore the short-term effects of application of reduced P fertilizer+manure on the network characteristics and stability of the AMF community in soil. [Results] The alpha diversity of AMF decreased in the case of application of reduced P fertilizer+manure compared with that in the case of inorganic P fertilization. Glomus and Paraglomus were the dominant taxa of AMF in each treatment. The average degree and average weighted degree of the network reached the maximums under the appropriate amount of P fertilizer, and were higher in inorganic P treatments than in reduced P fertilizer+manure treatments. The number of negative correlation links in the network increased with the increase in P application in inorganic P treatments and decreased with the increase in P application in reduced P fertilizer+manure treatments. The application of reduced P fertilizer+manure improved the stability of AMF community by increasing the negative/positive cohesion ratio. Compared with indicator species and keystone taxa, dominant taxa are closely associated with the stability of AMF community. [Conclusion] In acid purple soil, short-term application of reduced P fertilizer+manure regulated the alpha diversity and dominant taxa of AMF by changing soil pH, available P, and soil organic matter, thereby affecting the network complexity and stability of AMF community.

Abstract:The ecological effect of sulfate has attracted increasing attention, while little is known about the impact of sulfate on sediment microorganisms in aquaculture ponds. [Objective] To explore the influence rule and possible mechanism of different concentrations of sulfate on sediment microorganisms in aquaculture ponds. [Methods] The experimental systems were constructed with the sediment and surface water collected from an aquaculture pond. After the addition of 0 mg/L (control group), 30 mg/L (T1 group), 150 mg/L (T2 group), and 500 mg/L (T3 group) Na₂SO₄, the changes of the abundance, diversity, composition, and co-occurrence network of microbial community in the surface sediment were determined, and the environmental factors influencing the sediment microorganisms were analyzed. [Results] The sediment microbial communities showed little differences among groups within 30 days of incubation. On day 50, the microbial abundance and diversity in T2 and T3 groups decreased significantly compared with that of the control group. Compared with that in other groups, the relative abundance of Acidobacteriota and Bacteroidota in T1 group and that of Proteobacteria and Actinobacteriota in T3 group increased significantly (P<0.05). Compared with those in the control group, differential taxa increased in T1 group (62 taxa) and decreased in T3 group (45 taxa). The complexity of the microbial co-occurrence network was increased by high-concentration sulfate, which suggested that the microbial community might respond to the environmental changes caused by sulfate via self-regulation. The redundant analysis and correlation analysis revealed that the total organic carbon, total nitrogen, and oxidation-reduction potential of the sediment were the main environmental factors influencing the sediment microorganisms, suggesting that the sediment microorganisms might be affected by the interaction between sulfate and organic matter. [Conclusion] The long-time exposure to high concentrations of sulfate significantly affects the sediment microbial communities in aquaculture ponds, in which the transitions of microbial communities and changes in organic matter decomposition caused by sulfate may play a role.

Abstract:[Objective] To investigate the differentially expressed genes (DEGs) of Vibrio parahaemolyticus in the intermediate state of biofilm formation, and thus provide gene information for the future studies about the regulatory mechanisms of biofilm formation. [Methods] Illumina HiSeq and RNA sequencing (RNA-seq) assay were employed to analyze the gene expression of V. parahaemolyticus in the intermediate state of biofilm formation, and the results were then validated by quantitative real-time PCR (qPCR). [Results] A total of 979 DEGs were identified, including 379 down-regulated genes and 600 up-regulated genes. According to the results of gene ontology (GO) annotation, 363 DEGs were annotated to three functional categories (biological process, molecular function, and cellular component) and 30 sub-categories. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment showed that 706 DEGs were enriched in 37 pathways (Q value<0.05) and mainly involved in cellular metabolism and transport pathways. The results of clusters of orthologous groups (COG) classification showed that 888 DEGs were assigned to 20 categories, and the DEGs were mainly involved in amino acid transport and metabolism, general function prediction only, energy production and conversion, and function unknown. The expression trends of the DEGs validated by qPCR were consistent with the results of RNA-seq. In addition, the biofilm-associated genes and major virulence genes were identified from the RNA-seq data, including 10 c-di-GMP metabolism-associated genes, 1 lateral flagellar gene (lafA), 13 polar flagellar genes, 6 capsular polysaccharide synthesis genes, 6 exopolysaccharide synthesis genes, 5 type IV pilus synthesis genes, 6 membrane fusion protein (mfp) genes (cpsQ-mfpABC), 14 type III secretion system 1 (T3SS1) genes, 14 Vp-PAI genes (tdh2 and 13 T3SS2 genes), 3 type VI secretion system 1 (T6SS1) genes, 6 T6SS2 genes, 45 putative regulator genes, and 15 putative outer membrane protein genes. [Conclusion] A large number of genes demonstrate changed expression levels during the biofilm formation of V. parahaemolyticus, including the biofilm-associated genes, key virulence factor genes, and putative regulator genes. The data presented here provided important gene information for the future studies about the regulation of biofilm formation.

Abstract:Extracellular electron transfer (EET) of the electrochemically active microbe, Shewanella oneidensis, has promising prospects of application in pollutant degradation, environmental remediation, bioelectrochemical sensing, and energy utilization. The small tetrahaem cytochrome (CctA) is one of the most abundant proteins in the periplasmic space of S. oneidensis. Although CctA is involved in multiple redox processes, the knowledge on the behavior and mechanism of CctA in EET is limited. [Objective] To investigate the role of CctA in the EET with azo dyes as electron acceptors in the periplasmic space of the model strain S. oneidensis MR-1, and further enrich the knowledge about the mechanism of anaerobic respiration of S. oneidensis. [Methods] We took methyl orange (MO) as the electron acceptor to explore the characteristics of periplasmic MO reduction in ∆mtr (mtr-deleted strain) and investigate the role of CctA in EET by gene knockout and complementation. [Results] In the absence of Mtr complexes, CctA mediates the electron transfer for the periplasmic MO reduction. The reduction rate of MO had a positive correlation with the expression level of recombinant CctA at low concentrations, while higher levels of recombinant CctA did not further improve the reduction rate. The results of protein film voltammetry (PFV) distinguished CctA significantly from high-potential redox proteins in the periplasmic space, suggesting its involvement in a low-potential MO reduction pathway. [Conclusion] We uncovered the unique electron transfer behavior of CctA in periplasmic MO reduction from molecular dynamics, bringing valuable information for understanding EET, designing or modifying extracellular redox systems by means of synthetic biology, and facilitating the application of bioelectrochemistry in pollutant degradation.

Abstract:[Objective] The microorganisms with the function of heterotrophic nitrification-aerobic denitrification (HN-AD) play a key role in the biological removal of nitrogen, while little is known about the HN-AD microorganisms capable of simultaneous removing multiple inorganic nitrogen sources, especially hydroxylamine, from wastewater. In this study, a HN-AD strain EN-F4 capable of simultaneous removing hydroxylamine and nitrite was isolated from a vegetable field. We explored its nitrogen removal characteristics and the effects of hydroxylamine on the nitrogen conversion process, aiming to lay a foundation for improving the efficiency of wastewater treatment. [Methods] The strain EN-F4 was identified based on morphological characteristics and 16S rRNA gene sequence. Batch experiments were carried out to determine the nitrogen conversion characteristics of the strain EN-F4. The nitrogen balance, enzyme activity, and specific enzyme inhibitor tests were conducted to decipher the HN-AD mechanism of the strain EN-F4. Finally, the effects of hydroxylamine addition on the conversion of different nitrogen sources were explored. [Results] The isolate EN-F4 was identified as Pseudomonas oryzihabitans. The strain cultured at 25℃ showed the ammonium, hydroxylamine, nitrite, and nitrate removal efficiencies of 99.27%, 99.13%, 87.01%, and 85.20% and the maximum nitrogen removal rates of 8.27, 1.85, 5.10, and 5.31 mg/(L·h), respectively. The addition of hydroxylamine did not inhibit the denitrifying ability of the strain EN-F4, while it promoted the removal of nitrite and total nitrogen by the strain, with the maximum removal rates reaching 7.80 and 7.51 mg/(L·h), respectively. The enzyme activity, nitrogen balance, and specific enzyme inhibitors confirmed the strong HN-AD capability of the strain. [Conclusion] Pseudomonas oryzihabitans EN-F4 could efficiently remove inorganic nitrogen sources from wastewater by HN-AD at 25℃, and hydroxylamine could significantly promote the removal of nitrite and total nitrogen.

Abstract:[Objective] To investigate the small non-coding RNAs (sRNAs) in Corynebacterium glutamicum at different growth stages.[Methods] We examined the RNA-Seq data of C. glutamicum strains at different growth stages and transformed for industrial production and then employed sRNA-Detect and APERO approaches to construct the prospective sRNA library. real-time reverse transcription PCR (RT-PCR) was carried out to determine the expression of sRNAs. The functions, secondary structures, and conserved domains of potential sRNAs were predicted by bioinformatics tools. [Results] We constructed a library containing 2 653 potential sRNAs and performed classification and functional prediction based on their positions relative to the coding sequence (CDS) region. We identified sRNA00130 ubiquitous in Gram-positive bacteria with high GC content, sRNA00257 with varied expression during the growth period, and sRNA02036 and sRNA02037 with high expression. We predicted the possible binding sites and secondary structures of potential sRNAs based on free binding energy. The prediction results showed that potential sRNAs were associated with a variety of genes involved in cell replication and metabolic pathways. Most potential sRNAs were conserved only in C. glutamicum. [Conclusion] The discovery of potential sRNAs in C. glutamicum helps to fill the vacancy in the growth regulation mechanism of C. glutamicum and provides a new possible regulation tool and modification site.

Abstract:[Objective] To activate the silenced gene cluster for the biosynthesis of type II polyketides in Streptomyces cavourensis NA4 isolated from a marine environment, and identify the structure and antibacterial activity of the newly produced secondary metabolite. [Methods] The silenced or low-expressed biosynthetic gene cluster under laboratory culture conditions was activated by the addition of promoters and the knockout of negative regulator genes. The target compound was separated. The structure of the target compound was identified by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Furthermore, the antibacterial activity of the target compound was determined, and the biosynthetic pathway of the compound was deduced based on bioinformatics information. [Results] A biosynthetic gene cluster encoding type II polyketides was mined from the genome of S. cavourensis NA4 and successfully activated. A type II polyketide was separated from the expression products. The biosynthetic pathway of this compound was deduced and the antibacterial activity was determined. [Conclusion] Genome-oriented natural product mining can facilitate the separation of target products and fully tap the potential of Streptomyces in the production of secondary metabolites.

Abstract:[Objective] Bacillus subtilis, a Gram-positive bacterium ubiquitous in the nature, has strong stress tolerance and environmental adaptability and can inhibit the propagation of a variety of harmful bacteria. It is a common and important enzyme-producing bacterium, with the yield of proteases and amylases accounting for 50% of the industrial enzyme yield. Prophages integrated in host genomes are capable of providing additional genes and endow hosts with biological properties, demonstrating great research significance. The available studies about B. subtilis prophages focus on the defective ones. In this study, we analyzed the genome of an active prophage to expand the knowledge about non-defective prophages. [Methods] We induced a phage fromB. subtilis by using mitomycin C and named the phage as Bacillus phage Bsu-yong1. We employed transmission electron microscopy (TEM) to observe the negatively stained Bsu-yong1, Illumina MiSeq to sequence the genome of Bsu-yong1, and bioinformatics tools to annotate the genome. Furthermore, a proteomic tree of Bsu-yong1 was built based on genome-wide sequence alignment. [Results] Bsu-yong1 showed the morphology similar to that of PBSX-like defective prophages of B. subtilis. Unlike defective prophages which package DNA segments from random portions of the host genome, Bsu-yong1 was capable of packaging its own genome. The full-length genome of Bsu-yong1 was 43 590 bp with the G+C content of 41%. A total of 62 open reading frames (ORFs) were predicted with modular arrangement in the Bsu-yong1 genome. Bsu-yong1 harbored the ORFs encoding T7SS effector LXG polymorphic toxin, IMMA/IrrE and SMI1/KNR4. The former two were bacterial toxins and the later was an antitoxin. Toxins and antitoxins are members of the bacterial immune system, participating in bacterial competition and environmental adaptation. The gene encoding LXG polymorphic toxin in bacteriophages had never been reported before. A proteomic tree was built based on the whole genome alignment, revealing a long evolution distance between Bsu-yong1 and other phages. In the proteomic tree, Bsu-yong1 clustered with phages sv105, rho14, and vB_BteM-A9Y to form a monophyletic clade. These four phages all had PBSX-like morphology and shared 29 core genes. Pairwise sequence comparison (PASC) illustrated that the highest nucleotide sequence similarity between Bsu-yong1 and known phages was only 46.72%, which was much lower than the threshold (70%) to define a genus.[Conclusion] Bsu-yong1 represented a new unknown genus. We suggest the establishment of a novel family composed of Bsu-yong1, sv105, rho14, and vB_BteM-A9Y, which share 29 core genes. Bsu-yong1 carries immunity-related genes, which may benefit the host in bacterial competition and environmental adaptation. The findings enrich the bacteriophage gene database and enrich the knowledge of active Bacillus prophages.

Abstract:[Objective] Despite the extensive studies about the effects of Bacillus licheniformis on the immune response, disease resistance, and nutrition of Penaeus vannamei, little is known about the effects on the intestinal and environmental microbial communities of P. vannamei in the zero-water exchange aquaculture system. [Methods] The intestinal, seawater, and sediment samples were collected from the environment supplemented with B. licheniformis in food or water for 16S rRNA gene sequencing and linear discriminant analysis effect size (LEfSe). [Results] AddingB. licheniformis had little effect on the growth of P. vannamei, and different adding methods had little effect on the intestinal microflora. However, the addition of B. licheniformis changed the intestinal microbial community and improved the immunity of P. vannamei. [Conclusion] The findings help us to comprehensively understand the changes in shrimp intestine and environment after B. licheniformis is added in feed and water in the zero-water exchange aquaculture system, thereby providing basic information for choosing the right probiotics and addition ways to sustain the health of P. vannamei.

Abstract:[Objective] To understand the bacterial community structures, species composition, and diversity in different parts of the medicinal plant Capparis spinosa L., so as to provide a theoretical basis for developing the microbial resources in medicinal plants and revealing the interactions between microorganisms and hosts. [Methods] The aboveground tissue (fruit and stem) samples of plants and soil (rhizosphere soil and bulk soil) samples were collected. High-throughput sequencing of bacterial 16S rRNA gene hypervariable regions V5-V7 and V3-V4 was employed to compare the bacterial community structure, species composition, and function in different samples. [Results] A total of 3 649 (operational taxonomic unit, OTUs) were annotated for the sequencing results of the four samples, belonging to 1 051 genera, 464 families, 248 orders, 88 classes of 34 phyla. The bacterial diversity in soil samples was higher than that in plant samples, following the trend of rhizosphere soil>bulk soil>stems>fruits. The bacterial diversity in fruits was always the lowest and significantly lower than that in rhizosphere soil. At the phylum level, Proteobacteria was dominant in plant samples, Proteobacteria and Actinobacteriota in rhizosphere soil, and Firmicutes and Actinobacteriota in bulk soil. At the genus level, Achromobacter, Erwinia, Enterococcus, Exiguobacterium, Lactobacillus, and Klebsiella were dominant in the plant samples, and Planomicrobium,Kocuria, Paenibacillus, Streptomyces, Microvirga, and Arthrobacter in the soil samples. The beta diversity analysis indicated that the bacterial community structure had significant differences between plant samples and soil samples and was similar in the same type of samples. [Conclusion] The bacterial diversity and abundance in soil samples were higher than those in plant samples, and the bacterial community structure varied between different parts of Capparis spinosa L. The findings provide accurate microbial information for exploring the function of and utilizing the bacteria in this medicinal plant.

Abstract:[Objective] To reveal the epidemic state and transmission characteristics of foot-and-mouth disease (FMD) in China based on the data of FMD outbreaks during 2017-2022. [Methods] The spatial, temporal, and susceptible species distribution, the production chain distribution, and the molecular epidemiology and traceability of the epidemic strains in China were analyzed.[Results] A total of 54 FMD outbreaks were reported in 15 provinces between 2017 and 2022. The general situation of FMD in China was stable considering the zero outbreak of serotype Asia 1, no report of serotype A for consecutive four years (2019-2022), and sporadic occurrence of serotype O. During 2017-2022, 36 outbreaks of FMD were reported in cattle, one outbreak in sheep, and 17 outbreaks in pigs. There were five epidemic strains (O/Mya-98, O/Ind-2001, O/CATHAY, O/PanAsia, and A/Sea-97) simultaneously circulating in China, which were closely related to the FMDV strains in surrounding countries (e.g., Myanmar, Laos, and Vietnam) during this period. Epidemiological investigation suggested that the outbreaks (especially in cattle, which accounted for 66.7%) mainly occurred in long-distance transport (57%) and backyard (32%) with incomplete vaccination. [Conclusion] In the future, strengthening vaccination and preventing the introduction of foreign strains are still major measures for FMD prevention and control in China.

Abstract:There are abundant plant resources of Astragalus in the high-cold region of northwestern Yunnan. These plants have obvious rhizosphere effect and their rhizosphere microorganisms are considered medicinal resources with great antimicrobial potential. [Objective] To understand the species diversity of rhizosphere microorganisms of Astragalusforrestii growing in the special high-cold environment of northwestern Yunnan, and the chemical diversity and the antimicrobial and anti-biofilm activities of the secondary metabolites produced by cultivable strains. [Methods] Metagenomic sequencing and culture-dependent method were employed to explore the microbial diversity in the rhizosphere soil of A. forrestii. High performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) were combined with broth microdilution and microplate assays to evaluate the chemical diversity and antimicrobial and anti-biofilm activities of the ethyl acetate (EA) extracts of strain fermentation broths. [Results] The operational taxonomic units of the rhizosphere soil samples of A. forrestii were classified and annotated to 856 species belonging to 316 genera, 187 families, 105 orders, 54 classes, and 22 phyla. Bradyrhizobium was the dominant genus. A total of 95 cultivable strains of 54 species belonging to 27 genera were obtained, including 54 bacterial strains of 33 species belonging to 20 genera and 41 fungal strains of 21 species belonging to 7 genera, among which Bacillus and Penicillium were predominant. The secondary metabolites of the bacterial strain Pseudomonas tolaasii ZTB4 and the fungal strains Aspergillus tabacinus ZNF17, Lecanicillium aphanocladii ZNF15, and Umbelopsis nana ZTF31 displayed broad-spectrum antimicrobial activities. The secondary metabolites of strains ZTB4 and ZNF17 showed strong inhibitory activity against the biofilm formation of methicillin-resistantStaphylococcus aureus (MRSA). The main active components of these two strains were cyclic lipopeptides and flavonoids, respectively. [Conclusion] The rhizosphere soil of A. forrestii harbors rich microorganism, and the secondary metabolites of some cultivable strains have high chemical diversity and strong antimicrobial and anti-biofilm activities. The results provide a theoretical basis for the development and utilization of characteristic microbial medicinal resources in special environments in China.

Abstract:[Objective] To observe the effects of different concentrations of Qingyu Hejiang Decoction on reflux esophagitis (RE) and intestinal flora.[Methods] Thirty-six healthy male SD rats were randomly assigned into six groups. Except the sham operation group, the remaining five groups received anterior gastric ligation+external partial pyloric ligation for the modeling of RE. Two weeks after modeling, all the 30 surviving rats were randomly assigned into a sham operation group, a control group, a western medicine (pantoprazole sodium enteric-coated capsules+mosapride citrate dispersible tablets+compound lactobacillus acidophilus tablets) group, and high-, medium-, and low-dose Qingyu Hejiang Decoction groups (n=6). The rats in the sham operation group, the control group, and other groups were administrated with distilled water, distilled water, and corresponding drugs, respectively, by gavage. After 14 days of intervention, all the rats were sacrificed for the collection of tissue samples. The pathological changes of the esophageal tissue were observed via hematoxylin-eosin (HE) staining, and 16S rRNA gene high-throughput sequencing was employed to determine the bacterial composition in the intestinal mucosa. [Results] The RE rats showed significant changes and low diversity of intestinal flora. The control group showed decreased relative abundance of Firmicutes and Bacteroidetes, increased relative abundance of Proteobacteria, and increased Pseudomonas and Ralstonia. Low-, medium-, and high-dose Qingyu Hejiang Decoction increased the diversity of intestinal flora in RE rats. Specifically, they increased the relative abundance of Firmicutes and Bacteroidetes and reduced that of Proteobacteria. At the genus level, Qingyu Hejiang Decoction increased the relative abundance of probiotics such as Bacteroides, Lactobacillus, Ruminococcus,Oscillospira, Bifidobacterium, and Parabacteroides in the intestinal tract of RE rats. Moreover, the medium-dose group demonstrated the best performance, with the intestinal microflora diversity close to that of the sham operation group. Proteobacteria was the characteristic microorganisms in the control group, while Actinomycetes and Bacteroidetes were the characteristic microorganisms in the medium- and high-dose Qingyu Hejiang Decoction groups, which were the same as those in the western medicine group. [Conclusion] Qingyu Hejiang Decoction can effectively treat RE by reshaping the intestinal flora structure (reducing harmful bacteria, increasing probiotics, and increasing the diversity of intestinal flora).

Abstract:[Objective] To breed highly acid-tolerant strains of Oenococcus oeni and study their stress tolerance and malolactic fermentation (MLF) performance.[Methods] The mutagenesis of O. oeni SD-2a was carried out with the atmospheric and room temperature plasma (ARTP) method. The ethanol tolerance of the mutants and the MLF performance of the mutants in simulated wine and wine were investigated. [Results] After ARTP mutagenesis, five acid-tolerant mutants with high β-glucosidase activity were isolated by subculture under stress (pH 3.0). The mutants showed strong ethanol tolerance at high ethanol concentrations. Among them, the mutant ARTP-2 had the highest β-glucosidase activity and l-malic acid cumulative degradation in the simulated wine. Moreover, it demonstrated faster l-malic acid degradation rate in wine than O. oeni SD-2a and completed MLF on day 18. The total concentration of aroma components in the wine fermented by ARTP-2 was significantly higher than that by O. oeni SD-2a. [Conclusion] The mutant ARTP-2 had strong stress tolerance and MLF performance and played a positive role in wine aroma. The findings laid a foundation for the further development of high-quality commercial fermenters for MLF.

Abstract:[Objective] To explore gene expression profiles of Sparassis latifolia cultivated with different lignocellulose materials and provide a reference for mining the key genes and deciphering the mechanism of lignocellulose degradation by S. latifolia. [Methods] We examined the transcriptomes of S. latifolia cultivated in the liquid medium with pine, spruce, bagasse, fermented spruce, or fermented bagasse as the sole carbon source. Weighted gene co-expression network analysis (WGCNA) was performed on the gene expression of samples cultivated with different lignocellulose materials. [Results] There were only 20 differentially expressed genes (DEGs) between the spruce and pine groups, and 486 DEGs (the highest number) between bagasse and pine groups. Gene ontology (GO) enrichment analysis showed that the DEGs were mainly involved in oxidoreductase activity, monooxygenase activity, and iron ion binding. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly involved in pentose and glucuronate interconversions, methane metabolism, and glyoxylate and dicarboxylate metabolism. The expression of genes encoding glycoside hydrolases associated with cellulose or hemicellulose degradation was generally higher when the strain was cultured with fermented bagasse as the sole carbon source. The expression of genes encoding carbohydrate-active enzymes for lignin degradation or modification was generally higher when the strain was cultured with pine, spruce, or bagasse as the sole carbon source. A total of 10 co-expression modules were identified by WGCNA. Significant positive correlations existed between the green module and bagasse, between the blue module and fermented bagasse, and between magenta and turquoise modules and fermented spruce. GO enrichment analysis showed that the genes in the turquoise module were enriched in urea transmembrane transporter activity, methyltransferase activity, and monooxygenase activity, while those in the blue module in hydrolase activity and β-mannosidase activity. KEGG pathway enrichment analysis showed that the genes in the blue module were enriched in galactose metabolism, fructose and mannose metabolism, phenylalanine metabolism, arginine and proline metabolism, etc. From the interaction network, 12 hub genes were obtained, which may be involved in lignocellulose degradation or gene expression regulation. [Conclusion] Different lignocellulose materials significantly affected the gene expression profiles ofS. latifolia, which may imply the specific degradation strategies against different lignocellulose materials.

Abstract:[Objective] This study aims to analyze the structure and influencing factors of bacterial community in the rhizosphere soil of highland barley in different planting areas in Xizang, reveal the biological markers of rhizosphere bacteria in specific environments, and provide a reference for the exploration and research of rhizosphere growth-promoting bacteria and their roles. [Methods] We carried out 16S rRNA gene high-throughput sequencing and statistical data analysis to compare the composition and structures of the bacterial communities in the rhizosphere soils of highland barley cultivated in five cities of Xizang. We then analyzed the biological markers and the driving factors of community structure changes in the rhizosphere bacteria of highland barley. [Results] The sequencing of 45 rhizosphere soil samples yielded 10 715 operational taxonomic units (OTUs), which belonged to 2 783 species, 1 244 genera of 43 phyla. Actinobacteriota, Proteobacteria, Chloroflexi, Acidobacteriota,Bacteroidota, Firmicutes, Gemmatimonadota, Myxococcota, and Patescibacteria were the dominant bacterial phyla, with the relative abundance of 94.92%-96.56%. The community structure of rhizosphere bacteria showed significant differences among the five cities, with greater inter-group differences than intra-group differences (R=0.226 9, P=0.001). Actinobacteriota, Chloroflexi, Acidobacteriota, Bacteroidota, and Patescibacteria showed different relative abundance among the five cities (P<0.05). We identified potential biological markers in the rhizosphere soil of barley in all five cities. There were potential biological markers in the rhizosphere soil of highland barley in the five cities. Lasa and Shannan had only three and six unique bacterial clades, respectively, with more complex co-occurrence network and tighter connections between OTUs. The key phyla in the rhizosphere of highland barley were Proteobacteria, Chloroflexi,Actinobacteriota, and Acidobacteriota. Additionally, Endobacteria,Methylomirabilota, and Cyanobacteria were unique taxa in Linzhi, Rikaze, and Shannan, respectively. The changes in the bacterial community structure in the rhizosphere of highland barley were mainly related to environmental factors such as pH, total potassium, available potassium, carbon to phosphorus ratio, and altitude, with total potassium being the most important factor (r²=0.621 4, P=0.001). [Conclusion] The bacteria in rhizosphere soil of highland barley in Xizang have high diversity, with significant differences among the five cities. Additionally, different growing areas of highland barley have unique biological markers of bacteria in the rhizosphere. The findings provide a reference for revealing the role of unique rhizosphere bacteria in the growth and environmental adaptation of highland barley and for exploring excellent rhizosphere plant-promoting bacteria.

Abstract:[Objective] To investigate the effect and mechanism of Wickerhamomyces sp. KLBMP0506 in promoting the growth of Arabidopsis thaliana. [Methods] The strain KLBMP0506 was co-cultured with the wild-type A. thaliana in plates and inoculated in the A. thaliana plants cultivated in pots. The growth and physiological indexes of A. thaliana were determined, including fresh weight, dry weight, taproot length, number of lateral roots, chlorophyll content, and soluble sugar content. Further, qRT-PCR was performed on 11 selected genes involved in lateral root and taproot formation and auxin synthesis and transportation in A. thaliana. [Results] The inoculation with the target strain KLBMP0506 increased the fresh weight and lateral root number of A. thaliana in plates and the fresh weight, dry weight, stem length, chlorophyll content, and soluble sugar content of A. thaliana cultivated in pots. The partition plate test and analysis of the promotive substances in the fermentation broth of the strain KLBMP0506 showed that the volatile organic substances produced by the strain and the n-butanol and ethyl acetate extracts of the fermentation broth had significant growth-promoting effects on A. thaliana. In addition, after treatment with KLBMP0506, the expression levels of ABI4 and FLA1 involved in the lateral root formation were down-regulated, and those of AUX1, EIR1, and YUC4 involved in auxin synthesis and transportation were up-regulated. The results indicated that the strain KLBMP0506 may promote the growth of A. thaliana by regulating the expression of genes associated with lateral root formation and auxin synthesis and transport. [Conclusion] This study confirmed the growth-promoting effect of strain KLBMP0506 on the model plant A. thaliana, providing a theoretical basis for developing microbial fertilizers with the strain.

Abstract:[Objective] The clustered regularly interspaced short palindromic repeats/Cas9 nuclease (CRISPR/Cas9) system was employed to knock out the ubiquitin-specific protease 30 (USP30) gene from human embryonic kidney 293T (HEK-293T) cells, which established a cell model for studying the function of USP30 and the regulatory role of this protein in virus replication.[Methods] According to the sequence of the first exon in the overlapping region of different transcripts of USP30 in Ensemble, we designed two pairs of single guide RNAs (sgRNAs) and constructed the recombinant plasmids pX459-USP30-sgRNAs. The HEK-293T cells were transfected with pX459-USP30-sgRNA and treated with puromycin for the screening of transfection-positive cells, from which the monoclonal cells were screened out by limited dilution method. We then employed Western blotting and DNA sequencing to evaluate the knockout of USP30 and compare the replication of Senecavirus A (SVA) in the wild type and the cells with USP30 knockout. [Results] We confirmed that USP30 was knocked out from HEK-293T cells by Western blotting and DNA sequencing. Further experiments revealed that the replication level of SVA in the cells with USP30 knockout was significantly lower than that in the wild type, indicating a positive role of USP30 in SVA replication. [Conclusion] We successfully constructed the HEK-293T cells with USP30 knockout and confirmed that USP30 played a positive role in SVA replication. The constructed cell line provides a good cell model for further deciphering the mechanisms of USP30-associated immune response and SVA infection process and serves as a tool for studying the regulatory role of USP30 in viral replication.