Abstract:The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens human health and disturbs the social order. The development of vaccines, oral drugs, and antibodies against COVID-19 has played a positive role in controlling the spread of SARS-CoV-2 infections, reducing the disease severity and death risk. However, due to the large number of infected people and the rapid mutations of the virus, numerous variants that can evade the vaccines and neutralizing antibodies emerged and significantly weakened the protective effects of the vaccines and antibodies. Developing broad-spectrum neutralizing antibodies against SARS-CoV-2 or even pan-beta-coronaviruses is of great importance for the prevention and treatment of SARS-CoV-2 variants and other highly pathogenic beta-coronaviruses. This article systematically reviews the strategies for the screening and preparation, mechanisms of action, neutralization effects, and broad-spectrum properties of neutralizing antibodies against SARS-CoV-2, and discusses the current challenges and future research directions, aiming to provide a reference for the future research in this field.

Abstract:Antibiotics are considered one of the cornerstones of modern medicine. However, the abuse of antimicrobial agents, including antibiotics, has accelerated the emergence of super bacteria that can resist multiple antimicrobial agents. Resistance genes are the key factors leading to bacterial resistance and can be horizontally transmitted by mobile genetic elements (MGEs), such as plasmids, transposons, and insertion sequences, posing a serious threat to public health. In recent years, facing the outbreak of carbapenem-resistant and polymyxin-resistant bacteria, tigecycline has been regarded as the “last line of defense” against multiple drug-resistant bacterial infections in humans. Recently, a novel mobile efflux pump gene cluster tmexCD-toprJ, mainly existing in plasmids, was found. This gene cluster encodes resistance-nodulation-cell division (RND) efflux pumps, which can expel a variety of antibiotics, including tigecycline, out of bacteria, greatly enhancing the resistance of bacteria. The tmexCD-toprJ gene cluster has been horizontally transferred to humans, animals, and the environment by mobile elements such as plasmids, posing a serious threat to public health. However, research on the specific structure and functional mechanism of TMexCD-TOprJ remains to be carried out. This article systematically summarizes the distribution characteristics, transmission mechanisms, and efflux pump structures of tmexCD-toprJ and proposes measures to block its spread from the concept of One Health, offering a scientific basis for slowing down the dissemination of tmexCD-toprJ.

Abstract:China has abundant straw resources, with over 800 million tons of crop straw produced annually. Directly returning straw to farmlands or using it as fertilizer can not only reduce the use of chemical fertilizers and alleviate agricultural non-point source pollution but also achieve the recycling of crop straw. Lignin has a complex structure and is entangled with cellulose and hemicellulose. Therefore, in the natural decomposition process of straw, lignin is the main limiting factor. Mining lignin-degrading microorganisms and deciphering their degradation mechanisms to improve the degradation efficiency have gradually become research hotspots. By reviewing the available studies of lignin-degrading microorganisms, we compared the lignin degradation characteristics of fungi and bacteria and analyzed the advantages of composite degrading consortia. Subsequently, we summarized the properties of lignin-degrading enzymes and their expression characteristics in different microorganisms, and introduced the research progress in lignin degradation mechanisms and derived aromatic hydrocarbon metabolic pathways. Finally, we reviewed the application of lignin-degrading microorganisms in the production of straw fertilizers and put forward the application prospects and research directions of microbial degradation of lignin in straw.

Abstract:Transposons are important mobile genetic elements that mediate the spread of antimicrobial resistance. The transposon Tn7 is closely associated with antimicrobial resistance, carrying a transposition module and a class II integron. Tn7 encodes the transposition-associated proteins TnsABCDE for cut-and-paste transposition, and the transposition core machinery TnsABC can bind to triple-stranded DNA or Cas (clustered regularly interspaced short palindromic repeats associated)-RNA complexes to achieve transposition. In recent years, researchers have identified several novel Tn7 transposons mediating multidrug resistance, which play a role in mediating the acquisition and spread of bacterial genes conferring resistance to antibiotics, disinfectants, and heavy metals. In this paper, we review the genetic structure, transposition mechanism, prevalence of Tn7 transposons and novel Tn7 transposons mediating multidrug resistance in bacteria, with a view to providing a reference for the in-depth study of Tn7 transposons in bacteria.

Abstract:[Objective] To breed new Spirulina strains with low-medium temperature adaptability for expanding the geographical scope and time, increasing the yield, and reducing the cost of Spirulina cultivation. [Methods] The single cells or spheroplasts of Spirulina platensis ZJU0116 in large-scale cultivation were prepared by tissue homogenization and centrifugation. They were treated with 0.6% ethyl methane sulfonate for 30 min and 2.4 kGy ⁶⁰Co gamma ray, and then screened by low-temperature stress with 5 times of cold (12 ℃)/hot (38 ℃) abrupt alternation. The single filaments were cultured separately for the establishment of temperature adaptation surface, determination of protein content, and test of large-scale cultivation. [Results] We obtained a low-medium temperature adaptive mutant, which was named ZJU0116(LMTA), with the protein content equivalent to that of ZJU0116 and the temperature adaptation surface and average daily yield increased by 10.7% and 10.9%, respectively. ZJU0116(LMTA) had the helix number 54.4% lower, the filament length 42.1% lower, and the pitch 28.8% higher than its parent ZJU0116. Moreover, the randomly amplified polymorphic DNA (RAPD) analysis indicated that the amplification with the random primer S30 for ZJU0116(LMTA) produced an additional band at 470 bp, which revealed significant polymorphisms between ZJU0116(LMTA) and ZJU0116. [Conclusion] ZJU0116(LMTA) has good thermal adaptability and stable production, with a 10% increase in the dry algae powder output in large-scale cultivation. The new strain will promote the development of Spirulina industry towards a new stage of deep integration of the “dual carbon” goal and high-quality upgradation.

Abstract:[Objective] To investigate the rhizobial diversity of wild soybean (Glycine soja) in saline soil and screen out the salt-tolerant and plant growth-promoting strains for the cultivation of soybean. [Methods] We used the culture-dependent method to isolate rhizobia from wild soybean growing in coastal saline soil and then evaluated their growth-promoting characteristics and effects on wild soybean and cultivated soybean. [Results] A total of 87 rhizobial strains were isolated from the root and nodule samples of wild soybean, belonging to Sinorhizobium, Rhizobium, and Bradyrhizobium. Next, 24 strains of different species or from different samples were selected as representatives. Among the 24 strains, 16, 6, 16, and 6 strains had the abilities of producing indole-3-acetic acid (IAA), producing 1-amino-cyclopropane-1-carboxylic (ACC) deaminase, dissolving phosphorus, and producing siderophores, respectively. According to the above growth-promoting characteristics, we evaluated the growth-promoting and nodulation effects of 11 strains on wild soybean. The performance of Sinorhizobium americanus DL3 was superior to that of other strains. Finally, pot experiments were carried out to examine the effects of strain DL3 on the salt tolerance of wild soybean and cultivated soybean. Strain DL3 promoted the growth and lowered the level of proline in leaves, thus alleviating the salt stress of wild soybean and cultivated soybean. [Conclusion] Strain DL3 improves the salt tolerance of plants, which has important theoretical and practical significance for soybean cultivation in saline soil.

Abstract:[Objective] Phages are specific viruses that infect chassis cells in industrial fermentation. Due to the widespread existence and difficult eradication, phages greatly affect the yield of fermentation. The mining and functional verification of phage resistance genes can significantly enhance the anti-phage ability of chassis cells, so as to prevent phage pollution at the source. This study aims to mine the genes conferring the resistance to phages and construct phage-resistant strains. [Methods] Co-evolutionary screening, resequencing, recombinant strain construction, and phage infection experiments were carried out for the screening of the strains with phage resistance. [Results] Seven strains domesticated for resistance to phages were obtained through co-evolutionary screening. The genome resequencing and Annovar analysis identified mutations in 12 genes. Three genes with high mutation frequency, dnaE (DNA polymerase III subunit α): yhjH (cyclic diGMP phosphodiesterase): and rzoD (putative phage-lysed lipoprotein), were selected, and then the strains overexpressing the genes and the strains with knockout of the genes were constructed. The strains overexpressing the selected genes demonstrated obvious resistance to BL21 Virus 01, BL21 Virus 02, BL21 Virus 06, T1, and T7. The adsorption rates showed that dnaE and yhjH affected phage replication, while rzoD affected phage adsorption. Quantitative PCR was employed to further analyze the resistance of the strains with mutations of dnaE and yhjH to phages. The results showed that the mutations of dnaE and yhjH affected the replication process of BL21 Virus 01, while that of rzoD affected the adsorption process of BL21 Virus 01. [Conclusion] The mutations of dnaE, yhjH, and rzoD can resist phage infections, and the engineered strain BC11 with the mutations of all the three genes demonstrates a wide range of phage resistance. The findings provide a basis for the mining of phage resistance genes and references for the construction of strains with phage resistance.

Abstract:[Objective] By screening the inflammatory response system, we discovered that the African swine fever virus (ASFV) protein C717R induced an inflammatory response. This study aims to reveal the function of C717R protein by constructing recombinant C717R-lentivirus and observing the recombinant-induced inflammatory response in BALB/c mice. [Methods] C717R was screened out by the inflammasome expression system and used to construct the recombinant C717R-lentivirus. Mice were infected with the recombinant C717R-lentivirus to enable the expression of C717R in mouse tissue. Quantitative real-time PCR, western blotting, and enzyme-linked immunosorbent assay were employed to examine the package of C717R-lentivirus, protein expression, and the serum levels of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and interferon-beta (IFN-β), respectively. [Results] C717R protein was normally expressed in mouse tissue. The serum levels of TNF-α, IL-1β, IL-6, IFN-γ, and IFN-β were elevated in the expression of C717R protein in mice. The mRNA levels of C717R, TNF-α, IL-1β, and IL-6 were significantly up-regulated in the expression of C717R protein in mice. Western blotting demonstrated that expression of C717R protein induced the caspase-1 and IL-1β in different tissues. The lesions in the liver, heart, lungs, and other organs of BALB/c mice expressing C717R protein were severer than those in the control mice. [Conclusion] The expression of C717R protein induced an inflammatory response in BALB/c mice. The findings provide a basis for deciphering the mechanism of C717R protein-mediated inflammation.

Abstract:d-tagatose is a natural and rare ketohexose with blood glucose-lowering, anti-caries, gut microbiome-regulating, blood circulation-promoting, and anti-aging activities, and thus it is widely applied in food, pharmaceutical, and cosmetic industries. Though the enzymatic conversion of galactose into tagatose is a simple process, the high cost of galactose limits its industrial application. Fructose, an isomer of tagatose, is an economical alternative with stable supply. Through 4-epimerization, tagatose can be produced from fructose, which is an ideal substrate for tagatose production. [Objective] To discover new tagatose-4-epimerases essential for the efficient industrial production of tagatose. [Methods] Gene mining was performed to identify an unknown protein HCZ0 with 4-epimerization activity from Thermotogae bacterium. HCZ0 was heterologously expressed, purified, and characterized. [Results] HCZ0 had a molecular weight of 57.9 kDa and the specific activity of 0.9 U/mg. The enzyme showed the highest activity at 70 ℃ and pH 9.0, and it activity was significantly improved by 2 mmol/L Ni²⁺. HCZ0 exhibited the half-life of 180, 67, and 9 h at 60, 70, and 80 ℃, respectively. Under optimal conditions, the HCZ0 was able to catalyze 200 g/L fructose to produce 28 g/L tagatose within 2 h. [Conclusion] HCZ0 is an alkaline high-temperature enzyme with good thermal stability, which can provide inspiration for future theoretical research and industrial production.

Abstract:The phagocytosis rate of macrophages and the morphological changes of Candida albicans are the key points in the research on the host-pathogen interaction. [Objective] To construct C. albicans strains expressing the green fluorescent protein (GFP) or mCherry from the wild type SC5314, and use them to study the interaction between macrophages and C. albicans. [Methods] The effects of fluorescent protein expression on the growth, morphology, and virulence of the strains in mice were determined. Under the co-culture condition, flow cytometry and fluorescence microscopy were employed to determine the phagocytosis rate of macrophages and the morphological changes of C. albicans, respectively. [Results] We constructed C. albicans strains expressing GFP or mCherry. These engineered strains had consistent phenotype with the wild type and could be used for flow cytometry to determine the phagocytosis rate of macrophages and the morphological changes of C. albicans in co-culture. [Conclusion] The constructed strains expressing fluorescent proteins provided a new method for studying the interaction between macrophages and C. albicans.

Abstract:[Objective] To explore the vitamin B₁₂ synthesis in ruminant gastrointestine and evaluate the effect of Lactobacillus plantarum (LAC) or Macleaya cordata (MAC) on the synthesis of vitamin B₁₂ by the ileal microbiota of weaned goats. [Methods] Twenty weaned black goats with similar body weights and ages were randomly assigned into a control group (n=7), a LAC group (n=7), and a MAC group (n=6). The control group was fed with a normal diet, and the LAC and MAC groups with normal diets supplemented with LAC P-8 at 4.0×10⁹ CFU/g and 0.3 g/d MAC (3.75%), respectively. The contents in the middle section of ileum were collected for metagenomic sequencing. VB12Path and KEGG were employed to analyze the sequencing results. [Results] A total 55 and 49 key genes associated with vitamin B₁₂ synthesis were identified in VB12Path and KEGG, respectively. The alpha and beta diversity of genes involved in vitamin B₁₂ synthesis were changed by the supplementation of LAC or MAC (P<0.05). Compared with that in the control group, the diversity of the LAC group showed little difference and that of the MAC group decreased (P<0.05). The abundance of genes involved in precorrin-2 synthesis pathway, aerobic and anaerobic synthesis pathways, salvage pathway, and Post-AdoCbi-P pathway (e.g., gltX, cbiT, cobT, and btuD) in LAC and MAC groups was higher than that in the control group. [Conclusion] The supplementation of LAC or MAC enhanced the synthesis of vitamin B₁₂ by ileal microorganisms. This study expands our understanding and analytical method of the microbial vitamin B₁₂ synthesis.

Abstract:[Objective] Seeds are an important pathway for the intergenerational transmission of plant microbiota, while the systematic research on the microbiota carried by seeds remains to be carried out. In this study, we measured the bacterial abundance, determined the bacterial community structure, explored the effects of planting regions and rice cultivars on bacterial abundance and community structure, and identified the core bacterial taxa in rice seeds. [Methods] We collected 36 groups of samples of 18 rice cultivars, each from Hainan and Tianjin in China. Each group of samples contained 5 or 10 DNA samples, each of which was extracted from 3 seeds. The fluorescent quantitative PCR for bacterial 16S rDNA was employed to measure the bacterial abundance in seeds, and the factors influencing the abundance were explored. The 16S rDNA amplicon sequencing was performed to determine the bacterial community structure in seeds. Furthermore, bioinformatics tools were used to analyze the factors influencing bacterial community structure and the core bacterial taxa. [Results] We measured the bacterial abundance of 1 080 rice seeds and found that endophytic bacteria existed in the rice seeds after surface sterilization, with the average abundance of 1.53×10⁶ per gram of seeds. The bacterial abundance in seeds was affected by rice cultivars rather than planting regions. We measured the bacterial community structure of 180 amplicon libraries and found that the bacterial community in rice seeds was similar to that in rice plants, both dominated by Proteobacteria. Planting regions significantly affected the bacterial community structure in rice seeds. The rice seeds from different planting regions were clearly separated in principal co-ordinate analysis, while there was no significant difference between japonica and indica. In addition, rice seeds had core bacterial taxa, with the relative abundance up to 85.56%. [Conclusion] This study systematically revealed the bacterial abundance and community structure in rice seeds and their influencing factors, providing data and methods for using seed-borne microorganisms to improve rice health.

Abstract:[Objective] Echinochloa crusgalli is one of the major noxious weeds in China’s farmlands. The long-term use of chemical herbicides has caused eco-environmental problems, such as the enhanced weed resistance and environmental pollution. Therefore, it is urgent to develop environment-friendly high-performance herbicides. [Methods] The strain NX1 was identified by tissue isolation, morphological observation, and molecular biological methods, and its pathogenicity and influencing factors were studied by multivariate statistical analysis. [Results] The strain was probably a new species of Curvularia. It can grow and produce spores at 25-30 and pH 5-11, with the highest sporulation quantity at pH 9 and 30. Although light did not affect the mycelial growth, continuous light was conducive to sporulation of the strain NX1. The strain NX1 can grow with different carbon and nitrogen sources, with the best utilization of corn meal and sodium nitrate. The laboratory biocontrol experiments showed that humidity, leaf age, light duration, and spore concentration significantly affected the pathogenicity of NX1 strain, among which humidity was the key factor. The crop safety test showed that the conidial suspension of the strain was safe to rice, rape, capsicum, and eggplant. [Conclusion] The strain NX1 has low requirements for environmental and nutritional conditions and is suitable for industrial production and field application. Therefore, it has the potential to be developed into a bioherbicide.

Abstract:[Objective] To analyze the probiotic functions of Lactiplantibacillus plantarum HLPL03 derived from the stool of healthy newborns and evaluate the environmental tolerance and metabolism of functional oligosaccharides of this strain. [Methods] The tolerance of L. plantarum HLPL03 to extreme conditions was evaluated by treatments with the gastrointestinal environment, hydrogen peroxide, and antibiotics. The modified media were used to evaluate the metabolism of functional oligosaccharides by L. plantarum HLPL03. Furthermore, the effects of functional oligosaccharides on the antibacterial activities, hydrophobicity, and adhesion of L. plantarum HLPL03 were investigated. [Results] The viable count was above 10⁴ CFU/mL when L. plantarum HLPL03 was cultured at pH 2.5 for 3 h and close to 10⁷ CFU/mL after the strain was cultured in 0.30% bile salt for 6 h. The viable count increased significantly when L. plantarum HLPL03 was cultured with 1.0 mmol/L H₂O₂for 6 h (P<0.001). L. plantarum HLPL03 metabolized different functional oligosaccharides (except xylooligosaccharides) and inhibited common food-borne pathogens. Raffinose was the best oligosaccharide to improve the biological activity of L. plantarum HLPL03. It enhanced the surface hydrophobicity of the strain by 36.1% and increased the adhesion rate of the strain on Caco-2 cells from 16.78% to 42.11%. [Conclusion] L. plantarum HLPL03 from healthy newborns has good resistance to environmental stress, with the biological activities effectively promoted by functional oligosaccharides such as raffinose, serving as characteristic lactic acid bacteria for research and development.

Abstract:[Objective] Bovine viral diarrhea virus (BVDV) is the key pathogen that causes bovine viral diarrhea-mucosal disease. The structural protein Erns of BVDV can weaken the host immune defense at the initial stage of virus infection and induce an inflammatory response in cattle. Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome is an important member of the NLR family, regulating the occurrence and development of inflammatory diseases. The activated NLRP3 inflammasome can cause pyroptosis of host cells, thereby inducing cascading inflammatory responses. However, the molecular mechanism of the Ern protein in inducing an inflammatory response in BVDV infection remains unclear. [Methods] To explore the effect of Erns protein on the NLRP3 inflammasome-induced pyroptosis during BVDV infection, we constructed a eukaryotic expression plasmid pCMV-HA-Erns to overexpress the Ern protein of BVDV. The mRNA and protein levels of cysteine-dependent aspartate-specific protease 1 (caspase-1), apoptosis-associated speck-like protein containing CARD (ASC), NLRP3, and interleukin-1 beta (IL-1β) in BVDV-infected cells were determined. The gene expression and cleavage of gasdermin D (GSDMD) were examined. Furthermore, electron microscopy was employed to observe the bovine testis (BT) cells and examine the pyroptosis. [Results] The Erns protein significantly activated the NLRP3 inflammasome and caspase-1. The activated caspase-1 cleaved GSDMD to produce active GSDMD-N and create holes on the BT cell membrane for the contents releasing, thereby inducing pyroptosis. Furthermore, the activated caspase-1 cut pro-IL-1β to produce activated IL-1β in the BT cell supernatant. [Conclusion] We systematically analyze the role of BVDV Erns protein in activating NLRP3 inflammasome to mediate pyroptosis, which is vital for the development of vaccines and therapeutic drugs for the prevention of BVDV.

Abstract:[Objective] Shewanella baltica is a specific spoilage microorganism commonly detected in refrigerated seafood at the end of shelf life, while the knowledge about the roles of cold shock proteins (CSPs) in this bacterium is limited. This study aims to unravel the roles of three csp genes in S. baltica SB-19. [Methods] BEAST was employed to study the molecular evolutionary of csp genes in food-derived microorganisms of Gammaproteobacteria, and qPCR to determine the expression patterns of the three csp genes in S. baltica SB-19. The strains with csp gene knockout were constructed, and their growth rates, quorum sensing, spoilage ability were analyzed under different temperatures and environmental stresses. Finally, three strains with heterologous expression of csp were constructed, and their responses to different temperatures and environmental stresses were examined. [Results] Three csp genes, cspC, cspD, and cspG, were identified in S. baltica. All the cspD genes of Gammaproteobacteria formed a monophyletic clade and segregated from other csp genes approximately 1 109.6 million years ago (MYA). The cspC and cspG of S. baltica segregated approximately 858.8 MYA. Both cspC and cspG were involved in the response to cold shock, whereas cspD did not. The knockout and overexpression experiments revealed that cspG was essential for S. baltica survival at low temperature and played a universal role in the response to environmental stress, and cspC aided the bacterial response to stress. The cspD gene was associated with bacterial growth. Furthermore, the result of the experiments with inoculation of knockout strains in fish juice indicated that both cspC and cspG were associated with the spoilage ability of S. baltica below room temperature. [Conclusion] The three csp genes play different roles in S. baltica. The findings pave new ways for the research on the cold adaptation and spoilage mechanism of spoilage microorganisms.

Abstract:[Objective] To analyze the composition and metabolic characteristics of gut microbiota in Siberian crane (Grus leucogeranus) overwintering in the Poyang Lake. [Methods] The 16S rRNA gene high-throughput sequencing was employed to reveal the gut microbiota composition and PICRUSt to predict the function of Siberian crane overwintering in two artificial habitats (lotus field and paddy field) in the Poyang Lake. [Results] The alpha diversity indexes (Ace, Chao1, Shannon, and Simpson) showed that the gut microbiota diversity of Siberian crane in the paddy field were higher than those in the lotus field (P>0.05). The beta diversity based on Binary-Jaccard distance matrix revealed significant differences in the gut microbiota structure between the two artificial habitats (R²=0.312, P<0.05). The Siberian crane overwintering in different artificial habitats of the Poyang Lake showed varied gut microbiota composition. Terrisporobacter, Romboutsia, and Turicibacter were dominant genera in the gut of Siberian cranes in the lotus field, while Lactobacillus, Riemerella, and Catellicoccus in the paddy field. Line discriminant analysis effect size (LEfSe) analysis showed that the microorganisms like Lactobacillaceae, Lactobacillus, and Turicibacter with a strong ability to metabolize carbohydrates had significantly different relative abundance between the two artificial habitats. Functional prediction of PICRUSt showed that the intestinal microorganisms were mainly involved in metabolism, gene information processing, environmental information processing, and human diseases. At level 1, there were 5 types of metabolic pathways with significant differences (P<0.001). [Conclusion] Different species and number of birds and different food sources of the two artificial habitats resulted in significant differences in gut microbiota composition and characteristics of Siberian cranes overwintering in the Poyang Lake. PICRUSt functional prediction showed that there were significant differences in the abundance of functional genes of gut microbiota in the Siberian cranes overwintering in different artificial habitats, suggesting that Siberian cranes could adapt to the changes of food resources in artificial habitats by adjusting their gut microbiota. We preliminarily studied the composition and function of gut microbiota in the Siberian cranes overwintering in Poyang Lake. The findings underpin the relevant research and provide references for the conservation of overwintering Siberian cranes and the formulation of management measures.

Abstract:Exopolysaccharides (EPS) produced by microorganisms can promote the formation of large soil aggregates. EPS-producing strains have good application prospects in improving soil and promoting crop growth. [Objective] We isolated the bacteria with high yields of exopolysaccharides from soil samples and studied their soil-improving function, environmental adaptability, and broad-spectrum disease resistance, aiming to provide candidate strains for the preparation of soil-improving microbial agents. [Methods] The yield of EPS was determined by anthrone-sulfuric acid method. The taxonomic status of the strain was determined by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequencing. Soil culture experiments were carried out to evaluate the effect of the strain on the formation of soil aggregates.[Results] Three strains of bacteria with EPS yields greater than 500 mg/L were obtained. A-5 was identified as Bacillus licheniformis, XJ-3 as B. atrophaeus, and KW3-10 as B. halotolerans. After treatment with these strains, the content of soil macroaggregates (>0.25 mm) increased by 4.07, 2.14, and 3.16 times, respectively. The three strains exerted significant inhibitory effects on plant pathogens such as Streptomyces scabies, Fusarium oxysporum, Alternaria solani, and Rhizoctonia solani. They could tolerate the saline-alkali environment with pH 5-9 and NaCl content of 1%-9% and promoted plant growth. The content of indole acetic acid in the metabolites of KW3-10 was 25.58 mg/L. [Conclusion] Strains A-5, XJ-3, and KW3-10 can significantly promote the formation of soil aggregates and demonstrate broad-spectrum disease resistance and remarkable growth-promoting performance, serving as candidates for the preparation of efficient compound microbial agents.

Abstract:[Objective] To screen out the bacterial consortium capable of efficiently degrading pyridine and promote the degradation of high-concentration pyridine in wastewater. We screened out a pyridine-degrading bacterial consortium and studied the degradation characteristics and metabolic pathways of the consortium, aiming to provide a theoretical basis and technical support for the microbial degradation and complete mineralization of high-concentration pyridine in wastewater. [Methods] We used pyridine as the only nitrogen source to screen out an efficient pyridine-degrading bacterial consortium MD1 from the aerobic activated sludge and water samples of a pesticide wastewater treatment system. The 16S rRNA high-throughput sequencing was performed to reveal the bacterial species, diversity, and structure of MD1. Single-factor experiments were conducted to study the degradation characteristics of MD1. The metabolites of pyridine degradation by MD1 were initially identified by gas chromatography-mass spectrometry, and the possible degradation pathways of pyridine were deduced. [Results] The degradation rate of MD1 incubated at the optimal conditions of 30 ℃, pH 8.0, and 0.1% NaCl for 72 h reached 98.44%±0.27% when the initial concentration of pyridine was 1 400 mg/L. MD1 was mainly composed of Paracoccus, unclassified_Brucellaceae, and Achromobacter at the genus level. The results of metabolite detection suggested that the possible metabolic pathway of MD1 was pyridine→nicotinic acid→6-hydroxynicotinic acid→2,5-dihydroxypyridine→N-formylmaleamic acid→maleamic acid→maleic acid→CO₂+H₂O. [Conclusion] This study screened out a bacterial consortium MD1 with stable performance in degrading pyridine. The diversity, structure, and pyridine metabolic pathway of MD1 were analyzed, which enriched the resources of pyridine-degrading microorganisms.

Abstract:[Objective] To investigate the degradation characteristics and degradation paths of decabromodiphenyl ether (BDE-209) by complex bacteria communities based on six strains of BDE-209 degrading bacteria, so as to provide a scientific basis for the remediation of BDE-209-contaminated environments. [Methods] We determined the concentration of BDE-209 by high performance liquid chromatography (HPLC) and identified the degradation products by liquid chromatography-mass spectrometry. [Results] The combination of Brevibacillus sp. (M1) and Achromobacter sp. (M2) had the best degradation capacity for BDE-209. At 30 ℃, pH 7.0, and an inoculation amount of 15%, the combination showed the degradation efficiency of 87.7% for 10 mg/L BDE-209 after 120 h. The complex bacteria community M(1+2) degraded BDE-209 more efficiently and quickly than the single strains. The degradation efficiency of BDE-209 by M(1+2) increased with the increase in the initial concentration of BDE-209 within the range of 0.5-10 mg/L. Liquid chromatograph-mass spectrometer (LC-MS) identified 11 degradation products of BDE-209. M(1+2) degraded BDE-209 by debromination, hydroxylation, deprotonation, ether bond breaking, and ring opening. [Conclusion] The complex bacteria community M(1+2) demonstrates strong degradation capacity for BDE-209. The findings enriched the microbial resources for improving the bioremediation of BDE-209-contaminated environments.

Abstract:[Objective] To investigate the effect of hesperidin (Hes) and rosmarinic acid (RA) combination on the growth performance and the cecal morphology, antioxidant function, microbiota, and barrier function of finishing pigs. [Methods] Twenty-four 90-day-old Duroc×Landrace×Yorkshire male pigs were randomly assigned to control (Con), Hes, RA, and Hes×RA groups (n=6). The Con, Hes, RA, and Hes×RA groups were given a basal diet, a basal diet supplemented with 600 mg/kg Hes, a basal diet supplemented with 40 mg/kg RA, and a basal diet supplemented with 300 mg/kg Hes and 20 mg/kg RA, respectively. The trial period was 90 d and divided into two stages: early fattening stage (1-40 d) and late fattening stage (40-90 d). [Results] Compared with the Con group, Hes×RA increased the average daily feed intake in the early fattening stage, late fattening stage, and whole period, increased the average daily gain in the early fattening stage and whole period, and decreased the feed/gain ratio in the whole period (P<0.05). Hes×RA increased the mucosal thickness, total antioxidant capacity (T-AOC), and superoxide dismutase (SOD) activity and up-regulated the relative mRNA levels of nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) of cecum (P<0.05). The combination decreased the relative abundance of Proteobacteria, Terrisporobacter, Clostridium sensu stricto 1, and Romboutsia, increased the relative abundance of Bacteroidota,Lactobacillus, Muribaculaceae_norank, Phascolarctobacterium, and Rikenellaceae RC9, and increased the concentrations of isobutyrate and butyrate in the cecal digesta compared with the Con group (P<0.05). Furthermore, this combination up-regulated the relative mRNA levels of zonula occludens 1 (ZO-1), Claudin-1, and mucin-2 (MUC-2), reduced the content of interleukin-1β (IL-1β), and increased the content of secretory immunoglobulin A (SIgA) in the cecum (P<0.05) compared with the Con group. [Conclusion] The Hes×RA combination improved the growth performance and the cecal morphology, microbiota structure, antioxidant capacity, and barrier function of finishing pigs.

Abstract:[Objective] The endophytic fungus Colletotrichum gloeosporioides Cg01, isolated from Huperzia serrata, can produce huperzine A (HupA) with a low yield. As the strain was subcultured for generations, the yield decreased and the strain degraded. Studies have shown that epigenetic modification is associated with the synthesis of secondary metabolites. This study aims to increase the yield of HupA, improve the quality of degraded strains, and decipher the mechanism of secondary metabolite synthesis from the perspective of epigenetic modification.[Methods] The carbon sources and biotic elicitors for the rejuvenation media were screened according to the colony morphology, mycelial growth rate, biomass, and HupA yield. The histone methyltransferase inhibitors UNC0224 and BRD4770 were screened to improve the HupA yield. The expression of genes involved in epigenetic modification was determined. [Results] The addition of the water extract of H. serrata did not significantly affect the growth rate, morphological characteristics, or biomass of C. gloeosporioides Cg01, while it increased the yield of HupA, which was 1.67 times (125.7 μg/L) that of the control group in the fifth generation. Furthermore, the addition significantly down-regulated the expression of the histone methyltransferase gene Cg12377, the histone deacetylase gene Cg15620, and the DNA methyltransferase gene Cg02440, while it up-regulated the expression of the histone deacetylase gene Cg02312. UNC0224 had no significant effect on the HupA production, while 2-15 μmol/L BRD4770 increased the HupA yield (152.10-169.57 μg/L) and down-regulated the expression of Cg12377, Cg02440, Cg02312, and Cg15620. [Conclusion] The secondary metabolite production of C. gloeosporioides Cg01 could be maintained by adding the water extract of H. serrata. The addition of BRD4770, a histone methyltransferase inhibitor, could increase the yield of HupA. This study provides a reference for addressing the strain degradation during the large-scale production of endophytic fungi and studying the influence of histone methylation on the synthesis of secondary metabolites.

Abstract:[Objective] Medium-temperature Daqu is the saccharifying, fermentative, and aroma-producing agent used in the brewing of strong-flavor Baijiu. The current quality evaluation of medium-temperature Daqu mainly depends on sensory and physicochemical characteristics. Previous studies have employed amplicon sequencing to preliminarily explore the relationship between the quality grade of Daqu and the composition of microbial community. However, little is known about the impacts of microbial community functions on the quality of Daqu. [Methods] We employed metagenomics to analyze and compare microbial community composition and potential functions of medium-temperature Daqu with different quality grades. We then analyzed the activities and the gene abundance of key enzymes involved in lactate and acetate metabolism. [Results] The fungal community structure was different between two Daqu samples. Higher relative abundance of filamentous fungi, represented by Aspergillus (21.6%), Rasamsonia (6.8%), Paecilomyces (5.0%), and Talaromyces (4.4%), were observed in the first-grade Daqu (Grade_1). The relative abundance of genes involved in environmental information processing and cellular processes in Grade_1 was significantly higher than that in the second-grade Daqu (Grade_2). In Grade_1, Hymenobacter and Aspergillus contributed to the higher gene abundance of alpha-amylase, resulting in significantly higher liquefying activity. In Grade_2, Lichtheimia (11.8%), Rhizopus (13.4%), and Pichia (7.2%), and lactic acid bacteria such as Companilactobacillus, Weissella, and Limosilactobacillus had higher relative abundance. Moreover, the genes involved in metabolism such as carbohydrate metabolism, energy metabolism, and nucleotide metabolism had higher relative abundance in Grade_2. The more abundant lactic acid bacteria in Grade_2 was conducive to the higher gene abundance of alcohol dehydrogenase and Saccharopolyspora, Staphylococcus, Aspergillus, and Thermoactinomyces provided a higher gene abundance of carboxylesterase, enhancing the fermentative and esterifying activities, respectively. Furthermore, more lactic acid bacteria in Grade_2 might produce more lactate dehydrogenase to degrade lactic acid, and the higher gene abundance of acetate metabolism-related enzymes could promote acetate catabolism, which led to the significantly lower acidity in Grade_2 than in Grade_1. [Conclusion] We analyzed the microbial community functions of medium-temperature Daqu with different quality grades at the gene level and explored the potential microorganisms contributing to the differences in enzyme activities and acidity. The findings can underpin the establishment of a comprehensive Daqu quality evaluation system and the rational regulation of community functions.

Abstract:Mycoplankton play a key role in the marine food web and biogeochemical cycling. [Objective] To reveal the community structures of mycoplankton across different seasons and their key environmental drivers, we collected the surface water samples from seven stations in July (summer), September (autumn), and December (winter) 2017 and March (spring) 2018 in the Maowei Sea, Beibu Gulf, Guangxi Zhuang Autonomous Region. [Methods] We employed high-throughput sequencing of the internal transcribed spacer (ITS) gene to analyze the community structure and diversity of mycoplankton. [Results] The mycoplankton in the Maowei Sea belonged to 374 species, 282 genera, 167 families, 71 orders, 26 classes of 6 phyla. Ascomycota (82.14%) and Basidiomycota (10.74%) were the dominant phyla, with the total relative abundance of 92.88%. The Shannon and Simpson indices of the mycoplankton were the highest in spring, while the Chao1 and Richness indices were the highest in winter. Furthermore, the results of principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) showed that there was a significant difference in the beta diversity of mycoplankton community among different seasons (R=0.591 2, P<0.001). The random forest modeling analysis (RF) revealed that 20 species of mycoplankton including Aspergillus flavus, Gloeoporus dichrous, Aspergillus subversicolor, Whalleya microplaca, and Lentinus squarrosulus were sensitive to environmental changes and could be used to assess the eutrophication status of the Maowei Sea. The Spearman correlation analysis showed that the alpha diversity of mycoplankton was positively correlated with seawater pH, salinity, and dissolved oxygen (P<0.05) and negatively correlated with temperature, chlorophyll-a (Chl-a), and total organic carbon (P<0.05). The variance partitioning analysis (VPA) indicated that nutrients were the key factors affecting the community structure of mycoplankton. [Conclusion] This study provides a theoretical reference for revealing the diversity of marine mycoplankton in Beibu Gulf and a scientific basis for eco-environment monitoring and resource utilization in the Maowei Sea.

Abstract:[Objective] To establish an efficient transcription unit assembly system for the metabolic engineering of Sclerotium rolfsii. [Methods] Based on Golden Gate technology and Mobius assembly, the scaffold plasmids for DNA part domestication, single transcription unit assembly, and application plasmid (multi-transcription-unit) assembly, were designed and constructed to provide a complete multi-transcription-unit assembly system. [Results] Two Level 0 vectors for DNA part domestication, four Level 1 vectors for single transcription unit assembly, and four Level 2 vectors and 13 auxiliary plasmids for application plasmid assembly were constructed. Using this system, we assembled tens of plasmids for domesticated DNA parts, single transcription units, and function analysis of scleroglucan-related genes. The constructed application plasmids could be directly applied in Agrobacterium tumefaciens-mediated transformation, electroporation transformation, and protoplast transformation of S. rolfsii. [Conclusion] The plasmid system we built has strong capabilities of DNA design, assembly, and capacity, providing an efficient plasmid construction platform for metabolic engineering and functional genomics research of S. rolfsii in the future.