Abstract:

Abstract:[Objective] To explore the effect of water pollution stress on the bacterioplankton community in the river. [Methods] Principal component analysis was performed to classify the water pollution stress into high, intermediate, and low levels. The diversity, composition, and interaction of bacterioplankton in the main stream of Jinjiang River in Poyang Lake basin were analyzed via high-throughput sequencing technology. [Results] The dominant phyla of bacterioplankton in the Jinjiang River under pollution stress included Pseudomonadota (13.53%-45.83%), Actinobacteriota (17.05%-40.50%), Bacteroidota (5.79%-31.56%), Cyanobacteria (0.41%-59.31%), and Bacillota (0.11%-4.81%). Among them, the abundance of Bacillota differed significantly (P=0.03) between different levels of pollution stress. The bacterioplankton composition varied significantly (P=0.046) between different levels of pollution stress. The Chao1 and ACE indices were significantly higher in the areas with low pollution stress than in those with medium pollution stress (P=0.024 and 0.037), and Shannon diversity index did not show significant difference. The topological characteristics of the symbiotic network indicated that with decrease of pollution stress, the complexity and stability of the bacterial symbiotic network increased and the interaction between bacterioplankton enhanced. [Conclusion] Pollution stress significantly changed the structure and interaction of bacterioplankton in the Jinjiang River. This study provides ecological reference for the detection, monitoring, and control of water pollution from a microbial perspective.

Abstract:Nitrogen cycle driven by microorganisms plays an important role in the material cycle, purification of external pollutants, and maintenance of ecosystem balance in the mangrove ecosystems. Compared with other natural ecosystems, mangrove which inhabits in coastal saline or brackish water, boasts multiple nitrogen cycle processes and diverse related microorganisms which are affected and regulated by complex environmental factors. This study summarized the properties and characteristics of mangrove soil and reviewed the main nitrogen cycle processes driven by microorganisms in mangrove ecosystems, such as nitrogen fixation, nitrogen mineralization, nitrification, anaerobic ammonia oxidation, denitrification and dissimilatory nitrate reduction to ammonium. In addition, we discussed the coupling process of nitrogen cycle and other cycles. Finally, we analyzed the influence of environmental factors such as pH, salinity, season, crab activity, and mangrove species on the nitrogen cycle and the related microbial abundance and diversity in the mangrove ecosystems. This review is expected to serve as a reference for the protection and restoration of mangrove ecosystems.

Abstract:Iron, an essential microelement for phytoplankton growth, limits primary productivity in more than one third of the global oceans, especially in the high-nutrient low-chlorophyll (HNLC) sea areas. Ocean iron fertilization has long been considered as a geoengineering strategy to reduce the atmospheric carbon dioxide level. However, 13 experiments of artificial ocean iron fertilization (aOIF) demonstrated that the promotion effect of iron addition on carbon output in the deep ocean was significantly lower than expected. This paper summarizes the cycle of carbon in the ocean and the atmosphere, reviews the effects of artificial iron fertilization experiments on biological carbon pump and carbon flux, and analyzes the factors influencing the marine biogeochemical process from ocean iron fertilization to ocean carbon sink. According to the review, we conclude that the scientific community has limited understanding of the process and regulation mechanism of biological carbon pump. Considering the certain negative effects of ocean iron fertilization on the marine ecosystem, further research needs to be conducted to reveal whether iron fertilization can be adopted to reduce the carbon dioxide in the atmosphere to achieve carbon neutrality and mitigate the current greenhouse effect.

Abstract:Mangrove ecosystems are important coastal wetlands with highly efficient nutrient dynamics in the tropics and subtropics, and the hot zones driving the cycling of elements such as carbon, nitrogen, and sulfur. Sulfate-reducing prokaryotes (SRPs), one of the oldest microbial life forms on Earth, play a key role in driving early geological evolution and modern biogeochemical cycles of the Earth, while their role in mangrove wetlands remains to be studied. Only by fully understanding the processes and characteristics of the element biogeochemical cycles that SRPs participate in mangrove ecosystems can we capture the key aspects and clarify the coupling mechanisms. The development of bioinformatics has led to the discovery of more and more uncultured SRPs. In this paper, we systematically summarized the classification of sulfate-reducing bacteria and expand the taxa of SRPs based on the Genome Taxonomy Database prokaryotic genomes. We then introduced the distribution of SRPs in mangroves at home and abroad in recent years and the factors affecting the distribution to reveal the horizontal distribution diversity of SRPs in mangrove ecosystems. Further, we expounded the role of SRPs in the intertwined cycles of carbon, nitrogen, sulfur, and iron in mangrove wetlands, with emphasis on the molecular processes of SRPs in the sulfur cycle, especially dissimilatory sulfate reduction. Finally, we made an outlook on the future research directions of SRPs, aiming to provide references for further research on the element biogeochemical cycling and coupling driven by SRPs.

Abstract:Thaumarchaeota is an important microbial group in the global ocean, accounting for 20%-40% of the total marine prokaryotic plankton. As a group of chemautotrophs, Thaumarchaeota members are able to oxidize ammonia and leverage the released energy to fix inorganic carbon in the dark. They thus play a crucial role in the biogeochemical cycling of carbon and nitrogen. Thaumarchaeota are the major driver of ammonia oxidization in the ocean. The primary production by Thaumarchaeota through chemosynthesis serves as an important energy source for marine microorganisms, especially those in the deep-sea environments. With increasing efforts, the knowledge on the physiological and metabolic features of this group is expanded. This includes the evidence of heterotrophic metabolism, the discovery of thaumarchaeotal groups with no ammonia oxidation capacity in the deep sea, and the production of oxygen, nitrous oxide, and dinitrogen under anaerobic conditions. These studies reveal the new mechanisms of Thaumarchaeota in participating in the marine biogeochemical cycling and regulating climate change, which provide novel insights and perspectives for further studies and culture. In this study, we review new discoveries about marine Thaumarchaeota in recent years in terms of community composition, environmental adaptation, ecological function, evolutionary history, and culture status, with an aim of improving the understanding of this group.

Abstract:Ubiquitous microorganisms, key players in biogeochemical cycles and environmental evolution, are involved in environmental monitoring as well as ecological governance and protection. The booming high-throughput technologies have generated massive microbial data and expanded the scope of microbiome research. Constructing machine learning models to analyze complex microbial data is of great importance to microbial marker identification, pollutant prediction, and environmental quality prediction. Machine learning algorithms can be classified into two categories:supervised learning and unsupervised learning. In microbiome research, unsupervised learning grasps the characteristics of input data through clustering and dimensionality reductions, enabling the integration and classification of microbial data. Supervised learning uses microbial datasets with features and labels to train and build models that can be used to classify, identify, and predict new data without labels. However, sophisticated machine learning algorithms often focus on the accuracy of model predictions at the expense of interpretability. Machine learning models can often be regarded as a "black box" that predicts a specific outcome. Little is known about how the prediction is obtained by the model. Improving model interpretability is critical for the accurate application of machine learning and the extraction of valuable biological information in microbiome research. This review introduced the machine learning algorithms commonly used in environmental microbiology and the construction steps (including feature selection, algorithm selection, model construction and evaluation) of machine learning models based on microbiome data. Furthermore, we summarized several application scenarios of machine learning models in environmental microbiology for in-depth exploration of the relationship between the microbiome and the surrounding environment, attempting to improve the interpretability of the model and provide a reference for future environmental monitoring and environmental health prediction.

Abstract:Viruses are the most abundant biological entities on Earth. They play an important role in biogeochemical cycles and the evolution of life by regulating host community structure, causing mortality, and mediating genetic exchange. In recent years, the development of metagenomics has enabled the global-scale study of environmental viruses. A large number of novel viral genomes have been uncovered, and the roles and contributions of viruses in ecological processes and biogeochemical cycles have been recognized. The important role of viruses in ecosystem is mainly dependent on the infecting host. However, host prediction of environmental viruses has lagged far behind the studies of environmental viral genomics. This review aims to provide the latest knowledge of the main approaches for virus-host prediction, their pros and cons, and application, to reveal the importance of host prediction in the ecological research and bioengineering of viruses, and to present an outlook on the future development of host prediction approaches.

Abstract:Acid mine drainage (AMD) refers to acidic water that forms when sulfide minerals are exposed to air and water. AMD is generally characterized by low pH (<3.5) and high concentration of metal ions, which is thus one of the most serious threats to nature. Acidophilic prokaryotes, especially the iron- and sulfur-oxidizing bacteria and archaea that catalyze the generation of AMD, have long been the focus of research on acidophilic microorganisms. However, microbial eukaryotes in AMD environments often play a more important role in acidic ecosystems. Acidophilic photosynthetic algae are the primary producers and can drive the evolution and remediation of AMD. In this review, we outlined the research in recent 30 years on taxonomy and ecological distribution of acidophilic algae, mechanisms for their adaptation to the extreme environments, and the application in pollution control and industrial production. Finally, we proposed the future research directions of acidophilic algae which need to be strengthened.

Abstract:Glycerol dialkyl glycerol tetraethers (GDGTs) are the main components of membrane-spanning lipids in the cell membranes of archaea and some bacteria. As molecular fossils, GDGTs are sensitive to changes in environmental variables, and GDGT-based proxies have unique advantages in quantitative reconstructions of marine and terrestrial paleoenvironment studies. Although GDGT-based proxies have been widely applied in extensive studies, researchers have increasingly concerned about their fidelity and applicability. The key point lies in the lack of research on the biosynthetic and physiological mechanisms of GDGTs, which results in the deficiency of molecular biological and physiological basis for GDGT-based proxies. Recent breakthroughs achieved with interdisciplinary collaboration in the biosynthesis of archaeal isoprenoid GDGTs provide a reliable biological basis and a new vision for the implication of lipid biomarkers. In this paper, we present a holistic review on the biosynthetic pathway of archaeal isoprenoid GDGTs, propose a hypothetic biosynthetic pathway of bacterial branched GDGTs, and discuss the biogeochemical implications of the physiological process of GDGTs. Finally, we provide an outlook for future research on GDGTs.

Abstract:Polyphosphate (poly P) is a polymer of tens to hundreds of phosphate residues, which is found in various biological cells in granular, colloidal, and dissolved forms. It has been verified that poly P can be an energy source and regulate intracellular osmotic pressure to maintain the stability of membrane. In addition, it binds to protein and DNA to stabilize them and protects cells from stress injury. Granular poly P organelle stores poly P granules, metal cations, proteins, amino acids, and water in cells. The poly P granules in the parasite are called acidocalcisomes and those in bacteria or other microorganisms are named metachromatic granules. Further research shows that they have similar structures and thus they are both called granular poly P organelles. Crucially, this organelle is thought to be the only one preserved from prokaryotes to humans and may even be last universal common ancestor (LUCA). It is crucial for the origin of life, anti-stress, biological interaction, and metabolism regulation. It is meaningful to develop this organelle in pharmaceutical research and biogeochemical cycle of phosphorus.

Abstract:Human influenza is an acute respiratory infectious disease caused by influenza virus. Among the four types (A, B, C, and D) of influenza virus, influenza A and B viruses are the common pathogens causing human influenza. The patients with influenza virus infections mainly have respiratory syndromes and severe cases usually develop into pneumonia. In addition, the severe cases and mortality will increase in the case of coinfection or secondary infection of influenza virus with other pathogens (other viruses, bacteria, Mycoplasma, etc.). There has been an increasing trend of coinfections caused by influenza virus together with other pathogens. In this review, we briefly described the research progress in coinfections and secondary infections of influenza A or B virus with other pathogens, aiming to support the diagnosis and treatment of the complicated infections.

Abstract:As the intensive livestock breeding industry develops, veterinary antibiotics have been frequently used, allowing the emergence of antibiotic resistance genes in livestock manure. These genes pose great risks to ecological environment and human health. Thus, it has been a research hotspot to reduce antibiotic resistance genes in the livestock manure. This paper summarizes the source, distribution and influencing factors of antibiotic resistance genes in livestock manure, as well as the removal of antibiotic resistance genes from livestock manure by aerobic composting, anaerobic digestion, and their enhanced processes. Finally, in view of the problems in the available techniques, we summed up the future research directions, hoping to provide theoretical basis and technical support for the removal of antibiotic resistance genes in livestock manure.

Abstract:Amid economic and population growth, environmental pollution and water shortage have become problems faced around the world. Microalgae-based wastewater treatment can purify wastewater to tackle environmental pollution. In addition, the microalgae also use nutrients in wastewater to synthesize biomass. Thus, the technique has been popular. For the sake of high efficiency and low cost in wastewater treatment, it is necessary to clarify the mechanism by which microalgae remove nutrients and pollutants in wastewater, to develop low-cost downstream harvesting technology, and to improve the production of high-value by-products of microalgae. In this paper, the mechanism by which microalgae remove carbon, nitrogen, phosphorus, heavy metals, antibiotics, and organic matter and the influencing factors were reviewed, and the different harvesting methods of microalgae and the current applications of biomass in various fields were summarized. Finally, the advantages and disadvantages of different microalgal co-culture systems and microalgal immobilization techniques were analyzed, and the development of microalgae-based wastewater technology was predicted.

Abstract:Signal recognition particle (SRP)-dependent co-translational protein targeting is a conserved pathway across all kingdoms of life. It couples the translation and translocation of nascent peptides. Over 30% of newly synthesized peptides are delivered to the correct localization by SRP. Recent studies have demonstrated that SRP suppressors can circumvent the SRP requirement in Escherichia coli. The translational control plays a critical role in mediating membrane protein targeting when SRP is absent. This review summarizes how the substrates of SRP translocate to the proper localization with or without SRP, and how the decreased translation rate compensates for the loss of SRP. Furthermore, we discuss the dependence of different proteins on SRP. This review will provide new ideas for further studies about the function of SRP and membrane protein targeting.

Abstract:The discovery of aerobic denitrification has broken the traditional cognition that the denitrification only occurs under strictly anaerobic conditions. The aerobic denitrification provides a new way for biological denitrification and becomes a research hotspot in recent years. Carbon source can provide energy and electron donors for aerobic denitrification. The metabolism of carbon sources directly influences the nitrogen removal efficiency of aerobic denitrifying bacteria. Therefore, it is necessary to clarify the metabolic mechanism of carbon sources during aerobic denitrification. In this review, we summarized the known aerobic denitrifying bacteria and elaborated their metabolism pathways for nitrate and nitrite. Further, we systematically compared the mechanisms of different aerobic denitrifying bacteria in metabolizing carbon and nitrogen sources and comprehensively analyzed the effects of carbon metabolism differences on aerobic denitrification. Finally, we prospected the future research directions. With this review, we aim to deeply understand the mechanism of aerobic denitrifying bacteria in simultaneous removal of carbon and nitrogen and to provide a theoretical basis for improving the efficiencies of biological method for removing nitrogen and carbon from wastewater.

Abstract:Blood-brain barrier (BBB) is a natural structural and functional barrier, which can inhibit the entry of pathogens and control the entry of molecules into the brain parenchyma. An intact BBB is essential to maintain the homeostasis of the central nervous system. The function of this barrier is determined by its special multicellular structure. Each cell type has an indispensable contribution to the BBB integrity. Inflammasome known as a complex protein is a key component of the innate immune system and mediates the secretion of pro-inflammatory cytokines including IL-1β, IL-18, and IL-1α, which plays a critical role in the excessive inflammatory response. The up-regulated expression of proinflammatory cytokines results in the destruction of BBB. As a result, the pathogen breaks through BBB and enters the central nervous system, which finally causes brain diseases. In this paper, we reviewed infectious diseases and non-infectious diseases associated with the inflammasome-mediated injury of BBB.

Abstract:[Objective] To investigate the effect of straw returning method on physical and chemical properties and microbial community of black soil in Northeast China. [Methods] The experiment was carried out from December 2019 to October 2021. Two ways of straw returning were adopted:direct straw returning+microbial agent WJ (hereinafter referred to as MD) and straw composting before returning+microbial agent WJ (hereinafter referred to as MC). Soil fertility, enzyme activities, and microbial community were analyzed. [Results] The content of soil organic matter, humus and fulvic acid carbon was 2.28 g/kg, 7.82 g/kg and 5.26 g/kg, respectively, higher in MD than in MC. Content of soil ammonium nitrogen and available phosphorus in MD was slightly higher than that in MC, which peaked in June. Content of humic acid carbon decreased. In addition, the activities of urease, sucrase, cellulase and alkaline phosphatase in soil were 8.55%, 15.46%, 4.35% and 6.19% higher in MD than in MC, respectively. High-throughput sequencing results showed that the diversity of bacteria and fungi in MD was higher than that in MC. The dominant bacterial genera were Anaerolinea, Bacteroidetes and Pseudomonas, while Tausonia, Mrakia and Mrakiella dominated the fungi. [Conclusion] MD was conducive to the increase of soil organic matter, humus, enzyme activities and microbial diversity as compared with MC, suggesting that addition of WJ into straw can reduce the loss of organic nutrients and maintain the fertility of the soil in the field.

Abstract:[Objective] To analyze whether Monascus purpureus can improve the characteristics of sea buckthorn-highland barley complex Jiaosu. [Methods] The pH, total sugar, total acids, soluble solids, total phenols, total flavonoids, ascorbic acid and monacolin K, superoxide dismutase, lipase, protease, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging rate, 2,2ʹ-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid (ABTS) free radical scavenging rate, number of active yeast, and number of active lactobacilli were compared between control group, sea buckthorn group, sea buckthorn-highland barley Jiaosu group, and sea buckthorn-highland barley Jiaosu+M. purpureus group. [Results] The sea buckthorn-highland barley Jiaosu+M. purpureus group had higher total phenols, total flavonoids, ascorbic acid and monacolin K, superoxide dismutase, lipase, protease, DPPH free radical scavenging rate, ABTS free radical scavenging rate, number of active yeast, and number of active lactobacilli than the sea buckthorn group and the sea buckthorn-highland barley Jiaosu group (P<0.05). [Conclusion] The addition of M. purpureus can improve the characteristics of Jiaosu and plays a potential role in promoting the diversified development of the Jiaosu industry.

Abstract:[Objective] This study aims to investigate the effects of Fusobacterium nucleatum on the proliferation, adhesion, apoptosis, migration, invasion, and epithelial-to-mesenchymal transition (EMT) of colorectal cancer cell line HCT116 and human normal colon epithelial cell line HCoEpiC. [Methods] HCT116 and HCoEpiC cells were stimulated with F. nucleatum ATCC 23726 at different multiplicity of infection (MOI) to establish infection models, and then MTT, plate cloning assay, wound healing assay, and transwell assay were employed to measure cell proliferation, migration, and invasion. Flow cytometry was employed to detect cell apoptosis. The expression levels of E-cadherin, catenin δ-1, N-cadherin, and vimentin were determined by Western blotting. [Results] F. nucleatum promoted the proliferation, induced the migration and invasion, and did not induce the apoptosis of HCT116 cells. However, it inhibited the proliferation, migration, and invasion and accelerated the apoptosis of HCoEpiC cells. F. nucleatum exhibited strong adhesion to HCT116 and HCoEpiC cells, resulting in dispersed and elongated cells and reduced intercellular adhesion. This bacterium down-regulated the expression levels of the epithelial markers E-cadherin and catenin δ-1 and up-regulated those of the mesenchymal markers N-cadherin and vimentin in HCT116 and HCoEpiC cells. In addition, it promoted the transition of E-cadherin from the cell membrane to the cytoplasm. [Conclusion] F. nucleatum can adhere to both normal colon epithelial cells and colon cancer cells and induce EMT. However, it inhibited the proliferation, migration, and invasion of human normal colon epithelial cells, showing the effect opposite to that on colon cancer cells.

Abstract:As a major opportunistic pathogen, Serratia marcescens causes infections in human, animals, and plants, while it is rarely reported as a pathogenic bacterium of fish. [Objective] To study the pathogenicity of S. marcescens YP1 isolated from a diseased Japanese flounder (Paralichthys olivaceus) and the adverse effects of the pathogen on the fish disease control. [Methods] The morphological characteristics, molecular evidence, and physiochemical properties were combined to identify the strain YP1. Furthermore, the artificial infection experiment, histopathological examination, and drug sensitivity test of the strain YP1 were performed to study its infection symptoms, histopathology, virulence, and drug sensitivity. [Results] The strain YP1 isolated from the surface ulcer of a diseased Japanese flounder was identified to be S. marcescens. It had high pathogenicity to both Japanese flounder and zebrafish (Danio rerio), with LD₅₀ being 3.44×10⁷ CFU/g and 6.28×10⁵ CFU/g, respectively. YP1 mainly led to ascites fluid in Japanese flounder, which was accompanied by rapid breathing, reduced food intake, anal prolapse, white stool, gill ischemia, and multiple organ tumefaction and hemorrhage. With the extension of infection time, the organ damage became aggravated. The pathological sections showed that YP1 caused the damage of the gills, gut, liver, spleen, kidney, and heart in Japanese flounder. The results of drug sensitivity test showed that YP1 was sensitive to 14 antibiotics such as levofloxacin and norfloxacin and resistant to 19 antibiotics including ampicillin and cefradine. [Conclusion] This study confirmed that S. marcescens was a pathogenic bacterium causing ascites disease of Japanese flounder, and it was also highly pathogenic to other fish. Meanwhile, this study provides a scientific basis for the detection, identification, and prevention of the fish disease caused by S. marcescens.

Abstract:Virus infection leads to severe yield reduction and quality deterioration in garlic production. It is of great significance to collect, monitor, and conserve garlic germplasm resources for genetic improvement of garlic yield, virus resistance, and quality. [Objective] To reveal the virus-carrying status and distribution of garlic resources in China. [Methods] We employed two multiplex reverse transcription-polymerase chain reaction(RT-PCR) methods to evaluate the distribution of seven virus species among 689 garlic germplasm accessions. [Results]Allexivirus[GarV-A, GarV-B, GarV-C, GarV-D, GarV-E, GarV-X, and shallot virus X (ShVX)] had the highest detection rate (98.69%), followed by onion yellow dwarf virus (OYDV, 90.13%), garlic mite-borne filamentous virus (GMBFV, 71.12%), leek yellow stripe virus (LYSV, 50.65%), shallot latent virus (SLV, 49.20%), potato Y virus (PVY, 41.94%), and garlic common latent virus (GarCLV, 32.08%). Among these garlic accessions, 27 (3.92%), 122 (17.71%), 190 (27.58%), 150 (21.77%), 129 (18.72%), 62 (9.00%), and seven (1.02%) accessions carried seven, six, five, four, three, two, and one virus species, respectively, while two (0.29%) accessions did not carry any virus. In addition, there was a significantly positive correlation between the planting generations of garlic resources and the number of virus species (P<0.05). Shoot tip culture can effectively reduce or eliminate viruses. [Conclusion] This study reveals the distribution of seven main virus species in garlic germplasm resources in China, providing a theoretical basis for the safe preservation of garlic germplasm resources and enriching the gene resources for the breeding of antiviral garlic germplasm.

Abstract:[Objective] Soft rot of postharvest peach fruit, induced by Rhizopus stolonifer, causes huge economic loss. Synthetic fungicides, which are effective for controlling postharvest diseases of peach fruit, pose a threat to food safety, trigger resistance development in the pathogen, and pollute the environment in the instance of long-term use. In this study, we explored the potential inhibitory effect of the biogenic fungicide 1-octen-3-ol on soft rot of peach fruit, hoping to provide an environmentally friendly fungicide for this disease and a reference for the control of soft rot in postharvest peach fruit. [Methods] The peach fruit was fumigated with 1-octen-3-ol after the inoculation with R.stolonifer, and the expression of disease resistance-related genes and activities of related enzymes were determined. The effect of 1-octen-3-ol on the hyphae and spores of R.stolonifer was investigated in vitro. [Results] Fumigation with 1-octen-3-ol at 55.80 μg/mL reduced (P<0.05) the incidence of the disease and lesion diameter and improved the expression of nonexpressor of pathogenesis-related 1 (NPR1), pathogenesis-related protein 1 (PR1), chitinase (CHI), and β-1,3-glucanase (GLU) genes, and the activities of CHI and GLU. The in vitro experiment showed that 1-octen-3-ol could inhibit the growth of R.stolonifer hyphae on the plate and destroy the cell structure of mycelium. Moreover, 1-octen-3-ol could reduce the content of ergosterol in mycelium (P<0.05) and effectively suppress the germination of R.stolonifera sporangiospores and the elongation of germ tubes. It caused reactive oxygen species (ROS) burst and mitochondrial damage by destroying the membrane structure of spores, resulting in apoptosis. [Conclusion] 1-octen-3-ol can not only directly destroy the hyphae and spores of R.stolonifer, but also induce systematic acquired resistance (SAR) of peach fruit to inhibit the spread of postharvest soft rot.

Abstract:[Objective] Classical swine fever virus (CSFV) causes a highly contagious porcine disease that is characterized by hemorrhage syndrome and immunosuppression. In this paper, we studied the mechanism of CSFV inhibiting the host's innate immunity and its potential effect on influenza A virus (IAV) infection under immunosuppressive conditions. [Methods] First, the expression of interferons (IFNs) and IFN-stimulated genes (ISGs) and the phosphorylation of signal transducer and activator of transcription 1 (STAT1) induced by poly(I:C) in PK-15 cells which had been infected by CSFV were detected by reverse transcription-PCR (RT-PCR), reverse transcription-quantitative PCR (RT-qPCR), and Western blotting to explore the effect of CSFV on host innate immunity. Then, cells overexpressing CSFV proteins were used to screen and identify the key protein inhibiting the innate immunity. Finally, the potential influence of CSFV N^pro on innate immunity and IAV infection was examined in N^pro-overexpressing PK-15 cells by RT-PCR, RT-qPCR, Western blotting, and plaque assay. [Results] CSFV suppressed expression of type Ⅰ and type Ⅲ IFNs induced by poly(I:C). CSFV N^pro protein directly inhibited STAT1 phosphorylation and caused down-regulation of oligoadenylate synthase-like (OASL) protein, 2'-5'-oligoadenylate synthase-1 (OAS1), IFN-induced transmembrane protein 3 (IFITM3), and interferon-stimulated gene 15 (ISG15) in vitro. Moreover, CSFV N^pro protein suppressed the expression of type Ⅰ and type Ⅲ IFNs induced by IAV, and caused a dramatic decline in phosphorylation of STAT1 and expression of OASL, OAS1, IFITM3, and ISG15, thereby significantly promoting the replication of IAV in the N^pro-overexpressing PK-15 cells. [Conclusion] CSFV N^pro protein antagonizes the innate antiviral response induced by poly(I:C) and IAV, suggesting that N^pro protein can inhibit the RIG-I-dependent signaling pathway and promote IAV replication in the N^pro-expressing PK-15 cells.

Abstract:[Objective] To explore the expression, substrate degradation and thermal stability of Humicola insolens cutinase-OMP25 fusion protein (HiC-OMP25) in different Escherichia coli strains as well as the host cell membrane permeability and cell surface hydrophobicity, so as to reveal the differences in the expression of HiC-OMP25 by different host bacteria and further increase the expression of HiC-OMP25 in E. coli. [Methods] HiC-OMP25 was expressed in E.coli BL21(DE3) and E.coli C43(DE3), separately, and their degradation effect on 4-nitrophenol butyrate (pNPB) and polyethyl acrylate (PEA) and stability at 50℃ were determined. In addition, the changes in the cell membrane permeability and cell surface hydrophobicity of host bacteria were detected in HiC-OMP25 expression, and the expression of HiC-OMP25 in E.coli C43(DE3) was explored by co-expressing chaperone proteins. [Results] HiC-OMP25 was expressed in E.coli BL21(DE3) and E.coli C43(DE3) and pNPB was degraded. However, the degradation effect of the former on PEA and its stability at 50℃ were both lower than those of the latter. Additionally, HiC-OMP25 significantly enhanced the cell membrane permeability and cell surface hydrophobicity of E.coli BL21(DE3). Co-expression of HIC-OMP25 with sulfhydryl oxidase (Erv1p) and disulfide isomerase (DsbC) in E.coli C43(DE3) finally increased the expression level of HIC-OMP25 by 2.14 times, well degraded pNPB and PEA. [Conclusion] When heterologously expressed, HiC-OMP25 folded correctly in E.coli C43(DE3), but not in E.coli BL21(DE3). Co-expression of chaperone proteins improved the expression of HiC-OMP25 in E.coli C43(DE3), which laid a foundation for the industrial production and application of HiC-OMP25 in the future.

Abstract:[Objective] To obtain the new lasso peptide noncaromin, the biosynthetic gene cluster of noncaromin (nonc-BGC) from Nonomuraea candida HMC10^T was cloned and the heterologous expression in actinomycete classis strains as well as the antibacterial activity were explored. [Methods] The new nonc-BGC was initially defined by analyzing the whole genome sequence of N.candida HMC10^Twith antiSMASH. Then, complete nonc-BGC was cloned by ExoCET method from the genomic DNA of N.candida HMC10^T to obtain the recombinant plasmid pJQK652. The λ-Red recombination system was used to construct the integrative plasmid pJQK653, which, through conjugative manipulation, was introduced into Streptomyces albus J1074, S.lividans LJ1018, S.lividans 1326, S.coelicolor M1252, S.coelicolor M1452 and Saccharopolyspora erythraea LJ161 for heterologous expression. Subsequently target noncaromin was obtained by fermentation and purification. Finally, the chemical structure of noncaromin was determined by QTOF-ESI-MS², and its antibacterial activity was evaluated as well. [Results] In this study, complete nonc-BGC was cloned by EcoCET method, and its heterogeneous expression in six different actinomycete hosts were performed separately. Furthermore, we characterized the chemical structure of noncaromin and determined its negligible inhibition activity against Bacillus subtilis 168.[Conclusion] On the basis of the cloning of complete nonc-BGC from N.candida HMC10^T, the heterogeneous expression of nonc-BGC in six different actinomycete hosts was performed. As a result, a new noncaromin was discovered, with negligible inhibition activity against B.subtilis 168. These results provided reference for the discovery of new compounds in strain N.candida HMC10^T and other actinomycetes.

Abstract:[Objective] To explore a specific aminopeptidase that can debitter cod peptides and further increase the value of this fish. [Methods] Aspergillus oryzae RIB40-derived aminopeptidase (AoAPase) was expressed in Pichia pastoris KM71, and its characteristics were investigated. The cod peptides-debittering ability of AoAPase was evaluated by ANS probe and electronic tongue. The content and molecular weight of free amino acids in the cod peptides hydrolysate were detected by high performance liquid chromatography (HPLC), and the surface microstructure of cod peptides was observed under scanning electron microscope (SEM). Moreover, the DPPH-, hydroxyl- and ABTS-scavenging activities were determined to evaluate the antioxidant properties of cod peptides before and after AoAPase treatment. [Results] AoAPase was successfully expressed in KM71, with the molecular weight of about 41 kDa. AoAPase activity reached 2 238 U/mL under optimal conditions of 70℃ and pH 8.0. Its activity was enhanced by Ca²⁺. In addition, AoAPase exhibited substrate specificity, and its K_m and V_max values towards Leu-pNA were 5.95 mmol/L and 43.58 μmol/(mL·min), respectively. AoAPase could completely eliminate the bitterness of cod peptides by removing N-terminal hydrophobic amino acids and reducing the content of bitter peptides (500-1 000 Da). The cod peptide powder after AoAPase treatment became more delicate and loose with no significant change in antioxidant activity. [Conclusion] The recombinant AoAPase can remove the bitter taste of cod peptides without influencing their bioactivity. This study lays a theoretical foundation for the application of AoAPase in protein debittering.

Abstract:Endophytic actinomycetes of medicinal plants have the potential to synthesize natural active compounds, and new species of actinomycetes are an important source for discovering new compounds. [Objective] To explore the resources of endophytic actinomycetes of the medicinal plant Rehmannia glutinosa, and identify a new strain leaf-16 with the activity against the pathogenic fungus causing the ring rot of R. glutinosa. [Methods] In this study, we used five-step sterilization method to isolate the endophytic actinomycetes of R. glutinosa in Henan Province. Phoma herbarum, the pathogenic fungus causing the ring rot of R. glutinosa, was used as the indicator fungus to screen the antagonistic strains via the plate confrontation assay. For the antagonistic strains, 16S rRNA gene sequencing revealed a new actinomycete strain leaf-16, which was then identified based on the morphological, physiological, biochemical, and molecular characteristics, as well as cell wall components and DNA-DNA hybridization. [Results] We obtained eight actinomycete strains resistant to P. herbarum by plate confrontation assay. Among them, strain leaf-16 was identified as a new species of Streptomyces and named Streptomyces folium. [Conclusion] Strain leaf-16 is a new species of Streptomyces and has the activity of inhibiting the pathogenic fungus caused ring rot disease of R. glutinosa. This new species lays a material foundation for further isolation of new bioactive substances against the ring rot of R. glutinosa.

Abstract:[Objective] To develop a method of cloning biosynthetic gene clusters of natural products (NP-BGCs) by combining agarose-embedded chromosomal DNA strategy with exonuclease combined with RecET recombination (ExoCET) technology and transform the cloned gene clusters into chassis cells for the expression of target NP-BGCs in heterologous hosts.[Methods] Firstly, the chromosomal DNA of the targeted strain was prepared by agarose-embedded plugs with the low-melting-temperature agarose and digested with the restriction enzymes to yield the linear DNA sample. Then, the linear target BGC was captured by the linear vector of p15A through the ExoCET technology. The desired integrative and conjugative elements were introduced into the BGC-containing plasmid through PCR-targeting approach. Subsequently, the final modified plasmid was introduced into Streptomyces coelicolor M1252 by intergeneric conjugation to yield the desired recombinant strains. Finally, the recombinant strains were fermented and analyzed for target compound production by UPLC-ESI-MS and the inhibitory activity against different indicator strains was detected. [Results] With this method, the BGC of lincomycin (lmb-BGC) and the BGCs of two ribosomal peptides (nioblantin, niob-BGC and nitblantin, nitb-BGC) were obtained from S.lincolnensis NRR2936 and Nonomuraea nitratireducens WYY166^T, respectively. Finally, the lmb-BGC was expressed in M1252 for production of lincomycin. [Conclusion] In this study, the lmb-BGC and two novel lanthipeptide BGCs were cloned by the agarose-embedded chromosomal DNA in combination with ExoCET technology. Then, the BGC-containing plasmids were modified for conjugations. The recombinant strains MJX01, MJX02, and MJX03 were obtained by conjugation with the strain M1252 host. The fermentation broth was extracted and analyzed by UPLC-ESI-MS and the anti-bacterial activity was detected. Finally, our results revealed that the lincomycin was successfully produced in the strain M1252 containing the lmb-BGC. This study lays the foundation for the discovery of new compounds through gene cluster cloning and heterologous expression in the chassis strain.

Abstract:[Objective] To learn the prevalence and genotypic diversity of canine coronavirus (CCoV) in healthy and diarrhea dogs in Shandong Province. [Methods] We collected 199 anal swabs from healthy and diarrhea dogs in pet markets, stray dog centers, kennels, and pet hospitals in Shandong Province since 2017. CCoV was detected and genotyped by PCR method, and then the amplified M and S genes were sequenced and analyzed. [Results] Among the 199 anal swabs, 79 were CCoV-positive. The positive rate of CCoV was 40.2% (33/82) in healthy dogs and 39.3% (46/117) in diarrhea dogs. The positive rates of CCoV-Ⅰ and CCoV-Ⅱ were 78.8% (26/33) and 51.5% (17/33) in healthy dogs, and 39.13% (18/46) and 80.4% (37/46) in diarrhea dogs, respectively. CCOV-Ⅰ had the highest single infection rate (48.5%, 16/33) in healthy dogs, while CCoV-Ⅱ had the highest single infection rate (47.8%, 22/46) in diarrhea dogs. The M gene sequences of 48 strains were determined by sequencing analysis. Phylogenetic analysis showed that 11 out of the 12 CCoV-Ⅰ strains were isolated from healthy dogs. Among the 36 CCoV-Ⅱ strains, 7 strains were isolated from healthy dogs and the rest from diarrhea dogs. The 3 strains with whole S gene and obtained from diarrhea dogs all belonged to CCoV-Ⅱ.[Conclusion] The high positive rate of CCoV in the dogs in Shandong Province indicates that the virus is widely prevalent. Multiple infections exist in both healthy and diarrhea dogs. The main prevalent CCoV type is CCoV-I in healthy dogs and CCoV-Ⅱa in diarrhea dogs.

Abstract:brlA gene, as the initiator in the central regulatory pathway in Aspergillus conidiation, modulates the downstream conidiation-specific genes. Aspergillus without brlA fails to produce conidia, with growth and metabolism changed. However, the effect of brlA deletion is significantly different among different strains. [Objective] To explore the functions of brlM in Monascus, a homologous gene of brlA, and the regulatory mechanism of Monascus reproduction. [Methods] brlM was cloned from Monascus purpureus Mp-21. Based on homologous recombination, the brlM-deleted mutant (△brlM) was constructed by Agrobacterium tumefaciens-mediated transformation, and the colony phenotype, microstructure, growth rate, and secondary metabolites were compared between △brlM and the wild-type Mp-21 to clarify the functions of brlM in Monascus.[Results] For the morphology, △brlM showed more vigorous mycelial growth and a more fluffy phenotype of the colony than the wild type. According to the microscopy, △brlM lost the ability of producing cleistothecia through sexual reproduction but had stronger ability of produce conidia through asexual reproduction. Meanwhile, the yield of Monascus pigment, monacolin K, and citrinin of mutant △brlM was significantly lower than that of the wild type. [Conclusion] Different from brlA in Aspergillus, brlM gene is irreplaceable in the sexual reproduction of Monascus. This study provides a new idea for further research on the regulatory mechanism of the reproduction of filamentous fungi.

Abstract:[Objective] Apis mellifera ligustica is one of the subspecies of Apis mellifera. Ascosphaera apis exclusively infects bee larvae and leads to chalkbrood. In this study, we predicted the conserved motifs and analyzed the phylogenetic relationship of naked cuticle (Nkd) protein in A. mellifera. Further, we employed RNA interference (RNAi) to study the effect of nkd gene on the larval body weight of A. m. ligustica and the immune response to A. apis stress, aiming to enrich the information of A. mellifera nkd gene and reveal the role of nkd in A. m. ligustica larvae. [Methods] The conserved motifs of Nkd proteins from A. mellifera and nine other species were predicted by MEME. MEGA X was used for the phylogenetic analysis of these Nkd proteins. RNAi of the nkd in the guts of the worker larvae of A. m. ligustica was carried out by feeding dsRNA-nkd. The larvae were weighed via an electronic balance. RT-qPCR was employed to determine the relative expression of nkd and immune response genes. [Results] The nkd proteins from A. mellifera, Apis cerana, Papilio xuthus, Bombyx mori, and Papilio machaon contained three conserved motifs (motif 1, motif 2, and motif 3), which indicated that the Nkd proteins from the above-mentioned five insect species were highly conserved. The Nkd proteins in A. mellifera and A. cerana shared the same clade, showing the closest genetic relationship. Compared with the dsRNA-egfp group, the dsRNA-nkd group showed down-regulated expression of nkd in the guts of 5- and 6-day-old larvae (P<0.001), with the interference efficiencies of 49.60% and 56.40%, respectively. Additionally, the body weights of all the larvae in the dsRNA-nkd group were significantly lower than those in the dsRNA-egfp group. The expression of abaecin, apidaecin, birc5, defensin-1, and PGRP-S2 was activated in the guts of 4-day-old larvae. In the guts of 5-day-old larvae, the expression of abaecin, apidaecin, birc5, and defensin-1 was activated, while that of PGRP-S2 was inhibited. In the guts of 6-day-old larvae, the expression of abaecin was activated, while that of apidaecin, birc5, defensin-1, and PGRP-S2 was suppressed. The results suggested that the expression of above-mentioned five genes varied in response to stress and were involved in the host immune response. Moreover, there was a negative regulatory relationship between the expression of nkd and that of abaecin and apidaecin. [Conclusion] A. mellifera Nkd protein contains three conserved motifs (motif 1, motif 2, and motif 3). The Nkd proteins from A. mellifera and A. cerana shared the closest genetic relationship. The interference on nkd in the larval guts of A. m. ligustica workers could be achieved via feeding dsRNA, and nkd affected A. m. ligustica workers in terms of larval body weight and immune response to A. apis stress.

Abstract:[Objective] To investigate the inhibitory effects of 2-methylbutyric acid produced by Bacillus tequilensis XK29 on Ceratocystis fimbriata, and to evaluate its control effects on sweet potato black rot. [Methods] I-plates (2-section) and gas-phase antimicrobial system were used to study the inhibitory effects of 2-methylbutyric acid on mycelial growth and spore germination of C.fimbriata, and its effect on the micro-morphology of C.fimbriata was observed by lactophenol cotton blue staining. Fluorescent probes calcofluor white and propidium iodide were adopted to detect the influences of 2-methylbutyric acid on cell wall structure and cell membrane permeability of C.fimbriata, and the change of intracellular reactive oxygen species of C.fimbriata was explored by fluorescence probe 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate. Additionally, the content of glutathione was determined to analyze the ability of the pathogenic fungus to deal with oxidative damage, and the activity of mitochondrial dehydrogenase and the content of pyruvic acid were determined to clarify the effects of 2-methylbutyric acid on mitochondrial function and energy metabolism of C.fimbriata. In the end, we evaluated the application of 2-methylbutyric acid to the control of sweet potato black rot. [Results] 2-methylbutyric acid significantly inhibited the mycelial growth and spore germination of C.fimbriata, thus reducing the sporulation ability of C.fimbriata and resulting in folded mycelia and discontinuous cavities. The cell wall structure was changed, and the membrane permeability and the content of intracellular reactive oxygen species were increased. 2-methylbutyric acid markedly reduced the content of glutathione, thereby decreasing the ability of pathogens to deal with oxidative damages. Furthermore, the activity of mitochondrial dehydrogenase and the content of pyruvic acid dropped, which induced mitochondrial dysfunction and interfered with cell energy metabolism, thus leading to cell death. In addition, 2-methylbutyric acid controlled the sweet potato black rot.[Conclusion] 2-methylbutyric acid had remarkable inhibitory effects on C.fimbriata. It could be used as a safe and efficient gas-phase antimicrobial material for the development of new fumigants.

Abstract:[Objective] β-dimethylsulfoniopropionate (DMSP), an important sulfur-containing organic compound in the marine environment, is the main precursor to the climatically important gas dimethyl sulfide (DMS). DMS is released upon the bacterial catabolism of DMSP. The biological significance of this reaction is still unclear, and studies have shown that DMSP might be a chemoattractant. This study aims to identify the signal molecule in the chemotaxis of Alcaligenes faecalis J481 to DMSP and the receptor gene in this process. [Methods] All genes encoding methyl-accepting chemotaxis protein (MCP) were explored by genome mining, and the structures and functions of the chemotaxis receptors were predicted. The receptors-deleted mutants were developed by homologous recombination. The chemotactic phenotypes were observed based on the soft agar plate experiment and the chemotaxis receptors of DMS were determined. [Results] Through the test of DMSP, DMS, and acrylic acid, it was found that the signal molecule in the chemotaxis was DMS. A total of 8 potential genes encoding chemotaxis receptor proteins were found from the genome of J481, and D6I95_17420 was identified to encode the receptor protein of DMS. [Conclusion] D6I95_17420 encodes the chemotaxis receptor protein of DMS, which lays a foundation for further exploring the regulatory effect of DMS as a signal molecule in cells.

Abstract:[Objective] Vibrio parahaemolyticus is a significant foodborne pathogen that causes economic and public health problems worldwide. In this study, we explored the roles of the two-component system (TCS) EnvZ/OmpR in mediating the alkaline stress tolerance of V.parahaemolyticus.[Methods] We identified the two-component system EnvZ/OmpR in the V.parahaemolyticus chromosomes with SMART (https://smart.embl.de/). Then, we constructed the envZ-deleted strain, the ompR-deleted strain, as well as the complemented strains. On this basis, we compared the alkaline stress tolerance of the wild-type strain and the mutants, and the mechanisms whereby the EnvZ/OmpR regulated the alkaline stress tolerance of V.parahaemolyticus were explored by qRT-PCR and bioluminescence reporter assay. [Results] Homology analysis of the VP0155 and VP0154 proteins revealed that their amino acid sequences shared a high identity with the two-component system EnvZ/OmpR. ompR deletion decreased the tolerance of V.parahaemolyticus to osmotic stress and alkaline stress, while the growth status of C△ompR, △envZ, and C△envZ was similar to that of the wild type. The transcription levels of porin genes vp1218, vp0493, vpa1745, vpa0085, and vpa1308 in △ompR were lower than those in the wild type, and the deletion strains of the porin genes (except for △vpa1308) showed low growth ability in the alkaline-shock environment as compared with the wild strains. In addition, OmpR inhibited the expression of AphB and △aphB was significantly more tolerant to alkaline stress than the wild type. Moreover, AphB can directly repress the transcription of vp0493 and vpa0085. [Conclusion] The two-component system EnvZ/OmpR mediates alkaline stress tolerance of V. parahaemolyticus, which depends on the transcriptional factor AphB to regulate the expression of the porins.

Abstract:[Objective] To screen probiotics with inhibitory effect on murine colitis induced by Citrobacter rodentium and to investigate the mechanism. [Methods] The tolerance of the four screened strains was tested with simulated gastrointestinal fluid, and their inhibitory ability against C. rodentium was examined in vitro. Finally, Enterococcus faecalis MG 2108 was screened out. A total of 72 male 7-week-old ICR mice were randomized into two groups after 7-day adaptive feeding:normal control group (MC, 24 mice, normal saline) and inflammation control group (IC, 48 mice, 1×10¹⁰ CFU/mL C. rodentium). After 7 days, 12 mice were respectively selected from the two groups and the inflammation was examined based on hematoxylin and eosin (HE) staining of colonic tissues and detection of inflammatory factors. The remaining 12 mice from the MC were renamed as NC to distinguish the normal control group before and after modeling. The mice of IC group were randomly divided into three groups:natural recovery group (IR, 12 mice, normal saline), ciprofloxacin group (CF, 12 mice, 4 mg/mL ciprofloxacin), and E. faecalis MG 2108 group (EF, 12 mice, 1×10⁸ CFU/mL E. faecalis MG 2108). After 18 days of gavage, all mice were anesthetized and dissected and blood was taken from the eyes. [Results] E. faecalisMG 2108 alleviated the injury of colon and liver induced by C. rodentium. E. faecalis MG 2108 promoted the repair of colon tissue via decreasing the expression of inflammatory cytokines and increasing the expression of tight junction protein.E. faecalisMG 2108 induced structural changes in gut microbiota, as the abundance of beneficial flora such as Enterorhabdus and Akkermansia increased in EF group, and the content of short-chain fatty acids (SCFAs) in EF group was higher than that in CF and IR groups (P<0.05). [Conclusion] E. faecalisMG 2108, an intestinal probiotic strain, shows better effect on the C. rodentium-induced colitis than ciprofloxacin, and the effect of IR group was significantly inferior to that of EF group.