Abstract:In nature,most amino acids are encoded by more than one codon which is termed as synonymous codon.Along with the deep investigations,synonymous codon usage bias is considered as one important factor in gene replication,transcription,translation and modification.According to the biological functions of synonymous codon usage bias,the biased usage patterns have been verified in codon pair and codon co-occurrence as well.In gene expression,codon optimization can assist the gene of interest in enhancing its translational efficiency,which benefits the field of biological engineering in terms of protein expression.Moreover,synonymous codon usage pattern indirectly mediates biological processes related to transcription,chemical modification and translation in cells.The performances involved in the important life cycles mentioned above can be carried out by fine-tuning translation selection derived from synonymous codon usage bias.In this review,we tried to clarify how fine-tuning translation selection participated in gene expression and protein function via transcription,chemical modification and translation,and provide some new insights into optimal gene expression and the mechanism underlying the regulation of gene expression.

Abstract:The central nervous system and peripheral hormones jointly regulate the appetite of humans and animals.In recent years,it has been verified that the composition and changes of gut microbiota affect the host՚s appetite through multiple pathways.Intestinal bacteria can secrete and produce a large number of functional metabolites,such as short-chain fatty acids,secondary bile acids,and amino acid derivatives.In addition,gut microbiota can also affect the nutritional perception of the digestive system,delivery of intestinal vagus signals,secretion of intestinal hormones,among others,which are related to the appetite.Therefore,it is new strategy for improving host՚s appetite to clarify the mechanism of bacteria's regulation of appetite and perform targeted regulation and reorganization of gut microorganisms,which is helpful for the diagnosis and treatment of anorexia,bulimia,and other related diseases.

Abstract:The innate immunity can be considered as the first-line defense cascades that are gradually built up to maintain the host homeostasis in response to the intrinsic or extrinsic danger signals.The host cells have evolved multiple strategies to antagonize viral infections,and the innate immunity signaling pathways including interferon (IFN),nuclear factor-kappa B (NF-κB),and inflammasome will be activated when the pattern recognition receptors of the host cells sense pathogen-associated molecular patterns.IFNs are crucial to the antiviral immunity as they can induce the expression of IFN-stimulated genes to exert antiviral effects through various pathways.In addition,inflammatory response is an automatic defense response that induces the release of proinflammatory cytokines upon viral infection to regulate immune responses and exert antiviral activities.At the same time,IFN signaling pathway interacts with the inflammatory response regulatory network upon viral infections through some key molecules such as NF-κB/RelA and PKR.Furthermore,the IFN signaling pathway and the downstream cytokines can modulate the activation of other signaling pathways,which in turn regulate the immune response to maintain the homeostasis.An imbalance in the crosstalk can lead to excessive inflammatory responses,resulting in tissue injury.For example,the excessive inflammatory response induced by SARS-CoV-2 infection proves to cause tissue injury.In this review,we summarized the crosstalk between the IFN signaling pathway and the inflammatory responses upon viral infections,which was expected to provide insights into the future research on antiviral strategies.

Abstract:Infections of central nervous system (CNS) are acute or chronic inflammatory (or non-inflammatory) diseases caused by the invasion of viruses,bacteria,or fungi in CNS.Infectious diseases of CNS have high mortality rate with serious sequelae.Due to the limitation of detection strategies and low sensitivity,the pathogens of more than half of patients with infectious diseases of CNS cannot be identified by traditional methods.Metagenomic next-generation sequencing (mNGS) is a new technology and can significantly improve the detection rate of pathogens.However,mNGS is still not well understood by a few clinicians and other related people,which limits its rapid promotion and application in clinical diagnosis and treatment.This review systematically introduced the whole process of mNGS,and summarized the development history and the latest research progress of mNGS in the diagnosis of infectious diseases of CNS,thereby providing references for the diagnosis and treatment of infectious diseases of CNS.

Abstract:Food-borne pathogens threaten human life and health and antibiotics are the most effective choice at the moment.However,the irregular use of antibiotics has led to the rising prevalence of drug-resistant bacteria.Lactic acid bacteria are recognized as safe food-grade microorganisms.They boast good application prospects attributing to the functions of antagonizing pathogenic bacteria,improving immune regulation,strengthening intestinal barrier,and balancing intestinal microbiota.They are expected to become the next generation of safe,stable,and economical biological antibacterials to reduce or even replace antibiotics.This article describes the antibacterial substances,antibacterial mechanisms,and antibacterial functional properties of lactic acid bacteria to promote the research on and application of them.

Abstract:[Objective] Bacillus altitudinis 6ww6,a salt-tolerant and plant growth-promoting rhizobacterium,can be used as a preferred strain for microbial fertilizer.For protection of its intellectual property rights,it is essential to establish a method for rapid detection of this strain.[Methods] The orphan genes of strain 6ww6 were obtained through comparative genomic analysis and the elimination of homologous sequences by TBLASTn and NR library retrieval.The corresponding primers of the orphan genes were designed for PCR detection.[Results] Five specific genes (J939_13195,J9319_05960,J9319_13355,J9319_05965,and J9319_13350) were screened out for strain 6ww6.The target gene J9319_05960 of strain 6ww6 could be specifically amplified with primers 5960F and 5960R,which was confirmed to be the specific molecular marker of strain 6ww6.[Conclusion] In this study,we identified the unique identity code of strain 6ww6 and established a strain-level identification method based on comparative genomic and orphan genes analysis.This method can rapidly and accurately identify target species at the strain level,serving as a powerful tool for the protection of intellectual property rights of valuable microorganisms.

Abstract:[Objective]Cordycepic acid is one of the most important active substances in Samsoniella hepiali,but the low content greatly restricts its industrial application.Salicylic acid (SA) is an abiotic inducer,and we found that it could increase the content of cordycepic acid in S.hepiali.However,the metabolic pathways of cordycepic acid and its response to SA are not clear.This study was designed to obtain the transcriptome information of S.hepiali in response to SA treatment,and to explore key enzyme genes for the metabolic pathways of cordycepic acid.[Methods] In this study,SA was used to induce S.hepiali,and after 8 h treatment,high-throughput transcriptome sequencing was performed on induced and uninduced mycelia.[Results] A total of 40.37 Gb clean data was acquired,and 20 317 unigenes with an average length of 1 357.13 bp were constructed.Additionally,13 592 unigenes were annotated in the public databases NR,NT,KEGG,Swissprot,GO,and Pfam,and 2 574 differentially expressed genes were identified,in which 1 135 were up-regulated and 1 439 were down-regulated.KEGG enrichment analysis demonstrated that the differentially expressed genes were concentrated in the pathways such as cell cycle,meiosis,galactose metabolism,DNA replication,glycolipid biosynthesis,and glyceride metabolism.Furthermore,13 unigenes involved in metabolic pathways of cordycepic acid were found based on the assignment of KEGG pathway.The expression of genes glk,gpi,gla,mpi,fbp and mtld related to cordycepic acid biosynthesis was all up-regulated after the induction of SA,yet that of the gene mdh involved in cordycepic acid consumption was down-regulated,and the qRT-PCR results were in substantial agreement with RNA-Seq data.[Conclusion] Transcriptome information of S.hepiali induced by SA was obtained,and candidate genes for the metabolic pathways of cordycepic acid were identified.SA improved the expression of the biosynthetic genes and inhibited the gene transcription related to cordycepsic acid consumption,thus increasing the accumulation of cordycepic acid.This study provides reference for regulating the genes related to the biosynthetic pathways of cordycepic acid in S.hepiali by SA,and also facilitates the investigation on the regulatory mechanism of cordycepic acid metabolism.

Abstract:[Objective] Mycolicibacterium smegmatis was used to explore more efficient strategies for enriching phosphorylated peptides in prokaryotes.[Methods] We evaluated the efficiency of three different methods,TiO₂,Fe³⁺-NTA and Ti⁴⁺-IMAC,for the enrichment of phosphopeptides.Further,we employed two mass spectrometers with different resolutions,Orbitrap Velos and Orbitrap Fusion Lumos,to assess the enrichment stability.[Results] Ti⁴⁺-IMAC was the optimum enrichment method,with the number of phosphopeptides and sites enriched seven-fold that of TiO₂ or Fe³⁺-NTA.TiO₂ and Fe³⁺-NTA showed no significant difference in the number of phosphorylation sites enriched,which was similar to the results of the published works about TiO₂.In addition,the detection results of two different mass spectrometers showed that Ti⁴⁺-IMAC enrichment was stable in two biological duplicate samples.The average phosphorylation sites detected by Lumos was 2.6-fold that by Velos.[Conclusion]Ti⁴⁺-IMAC technique can efficiently accomplish high enrichment of phosphorylation events in M.smegmatis.We identified a total of 2 280 phosphorylated proteins,10 880 phosphorylated peptides and 4 433 credible phosphorylation sites.Ti⁴⁺-IMAC method can be widely used in the phosphoproteomics of other microorganisms.

Abstract:[Objective] Cyanobacterial blooms break out frequently and cause water quality deterioration.Cyanobacterial toxins (cyanotoxins) have hepatotoxicity,neurotoxicity,reproductive and genetic toxicity and tumor promoting effects,threatening the drinking water safety.Cyanobacterial blooms contribute to toxin accumulation or death of aquatic organisms,risking edible safety of aquatic products and causing huge economic losses to aquaculture industry.Cyanophages are viruses that specifically infect cyanobacteria and regulate its population density and abundance.They are considered as an important potential biological control tool for cyanobacterial blooms.Study on freshwater cyanophage is rarely reported,which is far less than that on seawater cyanophage.So far,little is known about Microcystis wesenbergii cyanophage.[Methods] In this study,we isolated a virulent freshwater cyanophage vB_MweS-yong2 by double-layer agar plate method using M.wesenbergii FACHB-1112 as the indicator host.Genome sequencing,open reading frame (ORF) annotation and phylogenetic analysis of cyanophage vB_MweS-yong2 were performed.[Results]vB_MweS-yong2 genome was a double-stranded DNA of 44 530 bp,with a G+C content of 71.6% and containing 61 ORFs and 1 tRNA gene.No vB_MweS-yong2 ORF was found to be associated with virulence factors and antibiotic resistance genes.The pairwise sequence comparison (PASC) scanning illustrated that the highest nucleotide sequence similarity between vB_MweS-yong2 and all known phages in databases was 20.21%,which was<50%(the boundary to define a genus).[Conclusion] vB_MweS-yong2 was proposed to represent a new genus in the Siphoviridae family of Caudovirales order.These works enriched the freshwater cyanophage database and laid a foundation for the research and development of the functional genes of cyanophages and the control of cyanobacterial blooms with M.wesenbergii as the dominant species.

Abstract:[Objective] Protein O-fucosyltransferase 1(POFUT1),a key enzyme catalyzing protein O-fucosylation,has been verified to regulate a series of physiological and pathological processes in animals and humans.However,POFUT1 gene in Colletotrichum fructicola or even fungi has not been reported.This study aims to clone the CfPOFUT1 gene from C.fructicola and analyze its biological functions.[Methods] RT-PCR was employed to amplify the CfPOFUT1 gene and then the bioinformation of the gene was analyzed.We constructed the CfPOFUT1 silencing and overexpression vectors,which were then transformed into the protoplasts with the PEG-mediated method to yield the CfPOFUT1-silenced and-overexpressing mutants.The biological phenotypes of the wild-type LY5-1,and the CfPOFUT1-silenced and-overexpressing mutants were determined,such as the mycelia growth,conidial production,conidial germination,appressorium formation,stress response,pathogenicity,and fungicide sensitivity.[Results]CfPOFUT1-overexpressing mutant showed higher conidial production,stronger pathogenicity,lower sensitivity to azoxystrobin,but higher sensitivity to carbendazim and prochloraz than the wild-type LY5-1.CfPOFUT1-silenced mutant demonstrated lower conidial production,intact cell wall,higher sensitivity to endoplasmic reticulum (ER) stress and azoxystrobin,lower pathogenicity,and lower sensitivity to carbendazim and prochloraz.[Conclusion] CfPOFUT1 is involved in conidial production,cell wall integrity,ER stress,and fungicide sensitivity of C.fructicola,affecting the pathogenicity.

Abstract:[Objective] The full-length cDNA infectious clone of jasmine virus C (JaVC),from Fujian Province was constructed.Coat protein (CP) genes of JaVC isolates from 9 provinces/autonomous regions in China were cloned and the motif difference was analyzed.Thereby,the distribution and transmission of JaVC in jasmine producing areas in China were investigated.[Methods] Total RNA of jasmine leaves which tested positive for JaVC was extracted and then reverse-transcribed to cDNA.The cDNA was then used as template for amplification to obtain the full-length genome sequence of JaVC,followed by the construction of full-length cDNA clone pXT-JaVC-FJ and the CP-fused red fluorescent protein mCherry clone pXT-JaVC CP-mCherry.Both of them were then transformed into Agrobacterium tumefaciens for Nicotiana benthamiana infiltration and verified through RT-PCR and confocal laser scanning electron microscopy.Samples collected from eight other provinces/autonomous regions of China positive for JaVC were then used for cloning and sequencing of the 3'end containing CP.Field investigation was conducted to identify the occurrence and transmission vector of JaVC.[Results] pXT-JaVC-FJ induced systemic infection on N.benthamiana by agro-infiltration,which demonstrated that it was biologically active.CPs of all isolates encoded 296 amino acids.The CP of isolate from Taiwan of China shared 82.27%–91.36% nucleotide sequences and 92.23%–96.82% amino acid sequences with those of isolates from the 9 provinces/autonomous regions.It showed the highest similarity to that from Guangdong and Yunnan Province at nucleotide level and amino acid level,respectively.The CP from different isolates mainly showed difference at aa 32‒35 and six motif arrangements of SEHA,GENA,REGT,SENA,GGDA,and GGNA were identified.Field investigation suggested that JaVC was widely distributed on jasmine plants and could be detected in thrips.[Conclusion]An infectious clone of JaVC-FJ is developed,which lays a foundation for the study of gene function and pathogenic mechanism of JaVC.The analysis of occurrence and variation of JaVC in indifferent jasmine producing areas in China is helpful for the prevention and control of JaVC diseases.

Abstract:[Objective] To explore the research status and development of actinomycetes and their metabolites.[Methods] We used CiteSpace for a bibliometric review of the literature extracted from Web of Science (WOS) during 2001-2021.[Results] China ranked the first in the world in terms of the number of the published papers involving actinomycete metabolites.Chinese Academy of Sciences had the largest number of published papers in this field among all institutions.[Conclusion] The research of actinomycete metabolites tends to involve multiple disciplines such as pharmacology,pharmacy,chemistry,biochemistry and molecular biology.In terms of the research content,most researchers focus on the biosynthesis of actinomycete metabolites.In addition,the mining of actinomycete germplasm resources has become a hot topic in the field.

Abstract:[Background] Streptomyces scabrisporus HBERC-53204 is an actinomycete strain isolated from a soil sample collected in Hunan province.It produces an active compound steffimycin B (SMB) with strong biological activity against a variety of animal and plant pathogens.[Objective] To improve the productivity of the strain for SMB and broaden the research and application of SMB in agriculture and animal husbandry.[Methods] We determined the optimum concentrations of main components including carbon-,nitrogen-containing nutrients and inorganic salts in the fermentation medium to increase the production of SMB by S. scabrisporus HBERC-53204 through single factor experiments.On the basis of the results of single factor experiments,the factors significantly affecting the production of SMB were screened out through Plackett-Burman (PB) design.On the basis of the steepest ascent design and Box-Behnken (BB) design,the medium for the production of SMB was optimized by response surface methodology through fitting the nonlinear equation of significant factors with the yield of SMB.[Results] The composition of the medium for the production of SMB by HBERC-53204 was optimized as follows:glucose 36.22 g/L,peptone 8.00 g/L,yeast powder 8.51 g/L,acid hydrolyzed casein 1.50 g/L,MgSO₄ 0.68 g/L,and KNO₃ 1.00 g/L.The yield of SMB produced in the optimized medium reached 477.26 mg/L in shake flask fermentation,which was 1 773.08% higher than that of the original medium.At the time point of 120 h of fermentation in a 20 L fermenter,the yield of SMB reached a maximum of 214.48 mg/L.[Conclusion] After optimization of the fermentation medium of strain HBERC-53204,the yield of SMB was significantly increased,which was scaled up in a 20 L fermenter.Based on this study,grams of purified SMB compound were obtained.

Abstract:[Objective] Oxytetracycline (OTC) is one of the first-generation tetracycline antibiotics,with good inhibitory effects on Gram-positive and Gram-negative bacteria.It is mainly used in animal husbandry and aquaculture industry,and also served as the raw material of second-generation and third-generation tetracyclic antibiotics.Therefore,there is an urgent need to improve productivity and reduce costs in production.To simplify genetic manipulation and shorten cultivation time,we evaluated the OTC biomanufacturing capacity of Streptomyces albus Del14 by heterologous expression and cluster refactoring.[Methods] Through rational cluster refactoring,we obtained a series of derivative strains:Del14:Oxy,Del14:Oxy1K,Del14:Oxy1KΔotrR and Del14B:Oxy1KΔotrR.The above strains were fermented in shake flask and OTC was analyzed by HPLC.Additionally,the transcriptional levels of structural genes related to otc cluster were detected by RT-qPCR.[Results] S.albus Del14 showed rapid growth and sufficient dispersed mycelia as well as high resistance to OTC.By manipulation of the expression of two cluster-situated regulators OtcR and OtrR,the OTC yield of the recombinant strain Del14B:Oxy1KΔotrR reached 1.1 g/L on the 6th-day of fermentation,which was comparable to the production by the native host S.rimosus M4018 on the 8th day.[Conclusion] In this study,the otc cluster was expressed in S.albus Del14 for the first time and the potential for OTC biomanufacture as a chassis strain was preliminarily confirmed,laying the foundation for further OTC production optimization.

Abstract:[Objective] Facility agriculture has experienced a surge in populairty in China which features a large population and little arable land.Greenhouse cultivation guarantees tomato security in China.However,greenhouse tomato are prone to gray mold (induced by Botrytis cinerea),leading to huge yield loss.Biocontrol is safe for human being and animal and environmentally friendly.It is urgent to seek more biocontrol microorganisms to improve the control effect.[Methods] In the current series of experiments,the biocontrol potentitial of a self-isolated new strain (LE16) of autolytic Lysobacter enzymogenes was explored.To be specific,pure culture was carried out to test the antagonism of LE16 fermentation broth against B.cinerea.Biochemical analysis,liquid chromatography-mass spectrometry,and transcriptome techniques were used to clarify the synthesis and secretion of extracellular hydrolases and antifungal compounds of LE16,and differentially expressed genes in B.cinerea,with a view to elucidating the antifungal mechanism.The inhibitory effect of LE16 fermentation broth on tomato gray mold was evaluated with the pot experiment in a greenhouse.[Results] LE16 synthesized and released siderophore,a variety of extracellular enzymes related to disease resistance (including phosphatase,protease,cellulase,β-1,3-glucanase and lysozyme),9 antifungal compounds (ustiloxin D,N-undecylbenzenesulfonic acid,rifabutin,nanaomycin,tigecycline,minocycline,m-cresol,cinnamic acid and cyclopentanone),and P-salicylic acid that is able to stimulate systemic acquired resistance in plants.The crude extract of LE16 fermentation broth significantly inhibited the growth and reproduction of B.cinerea,and the inhibition rate was 34.99%-100%,much higher than those of L.lysobacterium HYP18 and Ceriporia lacerata HG2011 on solid culture.Additionally,LE16 fermentation broth significantly induced the differential expression of genes in B.cinerea.The differentially expressed genes were involved in biological processes,cellular components,molecular functions (e.g.,protein synthesis,DNA replication and repair,and signal transduction),and metabolic pathways related to proteins,glycine,serine and threonine.In the greenhouse experiment,LE16 fermentation broth significantly enhanced the activity of antioxidant enzymes,including superoxide dismutase and peroxidase,in tomato leaves,and alleviated the damage to cell membranes caused by the pathogen.The inhibitory effect against gray mold varied from 72.54% to 74.42%,slightly lower than that of the chemical pesticide pyrimethanil.[Conclusion] LE16 suppresses the incidence of tomato gray mold in the greenhouse by multiple mechanisms,such as the synthesis and release of extracellular hydrolases,siderophore and antifungal compounds,and the induction of resistance to disease in the plants.Thus,LE16 shows a promising potential in controlling tomato gray mold in the greenhouse.

Abstract:[Objective] To investigate the effect of Legionella pneumophila recombinant immunogenic protein (IP) on RAW264.7 cell autophagy and the mechanism.[Methods] We purified the L.pneumophila IP using the His-tag protein purification kit and determined the half maximal inhibitory concentration (IC₅₀) of the IP on RAW264.7 cells with cell counting kit-8(CCK-8) assay.We co-cultured RAW264.7 cells with different concentration (0.05×IC₅₀,0.1×IC₅₀,0.2×IC₅₀) of IP for 1,3,6,and 12 h,respectively,and set up a cell control group.In addition,we used the pmCherry-GFP-LC3B tandem fluorescent protein to monitor changes in autophagy flux in RAW264.7 cells,and chose the optimal concentration for subsequent experiments.We co-cultured RAW264.7 cells with the optimal concentration of IP for 6,12,and 24 h,separately,and established a cell control group.Apart from that,the mRNA and protein expression levels of autophagy-related factors Beclin1,microtubule-associated protein 1 light chain 3B (LC3B),SQSTM1(sequestosome 1,p62),and histone deacetylase 6(HDAC6) were detected by RT-qPCR,Western blotting,and immunofluorescence staining.[Results] The IC₅₀ was calculated to be 0.26 μg/μL.When RAW264.7 cells were co-cultured with medium-concentration (0.026 μg/μL) IP,the autophagy flux was clearly inhibited,as monitored by pmCherry-C1-EGFP-LC3B.As revealed by the results of RT-qPCR and Western blotting analysis,when RAW264.7 cells were co-cultured with IP for 6 h,the expression level of P62 increased,but the expression levels of LC3B,HDAC6,and Beclin1 decreased (P<0.05).The expression of LC3B reduced and the expression of P62,HDAC6,and Beclin1 rose at 12 h compared with those at 6 h (P<0.05).Beclin1 expression was higher at 24 h than that at 12 h (P<0.05).According to the RT-qPCR and Western blotting results,IP decreased the LC3-Ⅱ/LC3-Ⅰ ratio and increased the expression level of P62 in a time-dependent manner (P<0.05).The immunofluorescence results were essentially consistent with the RT-qPCR and Western blotting results.[Conclusion] L.pneumophila recombinant IP can inhibit autophagy flux in macrophages.Moreover,IP may affect the autophagosome-lysosome fusion pathway and interfere with the formation and maturation of autophagosome and autophagolysosome.

Abstract:[Objective] Class Ⅰ lanthipeptides usually have a wide range of biological activities,with unique antibacterial mechanism and little drug resistance,which leads to the promising clinical applications.This work intended to explore two novel class Ⅰ lanthipeptides in Streptomyces coelicolor A3(2).[Methods] First,antiSMASH was used to analyze the S.coelicolor A3(2) genome sequence and mine lanthipeptide biosynthetic gene clusters.BLAST was employed to annotate gene function and select genes that might be involved in the biosynthesis process.Then,we constructed plasmids via DNA assembly and transferred them into Streptomyces chassis cells for heterologous expression.Finally,high performance liquid chromatography (HPLC),mass spectrometry (MS) and bioactivity assay were performed to detect the fermentation products.[Results] We reconstituted cluster 3(8.9 kb) and cluster 24(9.0 kb) of S.coelicolor A3(2) by adding promoter elements,and pYES-ColE1-SCO-cluster3 and pYES-ColE1-SCO-cluster24 were obtained.pYES-ColE1-SCO-cluster3 was successfully expressed in S.coelicolor M1152 and Streptomyces sp.A14 separately,and the potential target compound coelin 3 was obtained.pYES-ColE1-SCO-cluster24 was heterologously expressed in Streptomyces sp.ZM13,and the potential target compound coelin 24 was obtained.Coelin 3 exerted inhibitory effects on Bacillus subtilis 168 and Escherichia coli ATCC 25922,with the inhibition zone reaching 28 mm.[Conclusion] This study realized the expression of coelin 3 and coelin 24 and conducted the bioactivity assay by promoter activation and heterologous expression,which laid a foundation for subsequent structural and mechanism analysis of the novel class Ⅰ lanthipeptides.

Abstract:[Objective] This study aimed to construct a synthetic microbiome which would be used as a starter for Pixian Douban (broad bean paste) fermentation.[Methods] We analyzed core microorganisms of Pixian Douban fermentation by the combined indicators,including microbial relative abundance,frequency and eigenvector centrality.Then a fully synthetic medium which simulated the in situ fermentation system was designed and used to explore the growth and aroma-producing characteristics of core microorganisms.According to the complementary aroma-producing characteristics of core microorganisms,a two-strain co-fermentation experiment was conducted.Moreover,taking the microbial interactions into account,we designed a three-strain community and verified its fermentation performance.[Results] In this study,9 microorganisms were identified and isolated from Pixian Douban as the core microorganisms.Then the volatile profiles of these core microorganisms were detected and a complementary aroma-producing relationship was found among yeasts,lactic acid bacteria and other microorganisms.Combining the growth inhibition relationship among microorganisms,we formed a three-strain combined inoculum,including Pediococcus acidilactici,Staphylococcus carnosus and Candida versatilis.With the three-strain community as the fermentation starter,the fermentation samples showed 63.1% similarity of flavor compounds and 21.8% improvement of amino acid nitrogen content compared to the in situ samples.[Conclusion] A bottom-up strategy for constructing a synthetic microbiome community was proposed,and a three-strain microbial community as the starter for Pixian Douban production was built.This study is valuable for synthetic microbiome construction and fermentation process optimization.

Abstract:[Objective] Paracoccus denitrificans is an environment-friendly strain of α-Proteobacteria.It can also perform denitrification under aerobic conditions and has good denitrification ability.This study intended to use P.denitrificans DYTN-1 as the chassis cell to screen nitrogen inducible promoters for strengthening the nitrification and denitrification pathways,thus achieving the purpose of enhancing the removal of nitrogen pollutants by metabolic engineering.[Methods] Recombinant plasmids overexpressing amoA,amoB,hao and nirS genes were introduced into P.denitrificans DYTN-1 cells by conjugation.The gene elements and nitrogen removal ability of P.denitrificans DYTN-1 were characterized by fluorescence quantitative detection and nitrogen quantitative detection.[Results] Six promoters induced by NO₂^‒,NO₃^‒ and NH₄⁺ were obtained at 2 to 26-fold induction.The residual amount of NO₃⁻ in the medium of the nirS overexpressed strain was 67% of that of the wild-type strain after treatment with 2 g/L KNO₃for 24 h.The strains overexpressing both hao and nirS had only 50% of the residual NO₃⁻ of the wild-type strain after treatment with 1 g/L NH₄Cl and 2 g/L KNO₃ for 12 h.Furthermore,the final degradation efficiency of total nitrogen reached 79.5% and the residual total nitrogen was only half of that of the wild-type strain.[Conclusion] It is feasible to carry out metabolic engineering with the aboved screened promoters to enhance the removal of nitrogen pollutants in P.denitrificans DYTN-1.

Abstract:[Objective] Corynebacterium pseudotuberculosis(Cp) infection of animals often causes visceral and superficial lymph node abscesses,which is characterized by a large increase in inflammatory cytokines at the infected site. This study aimed to investigate the effect of Corynebacterium pseudotuberculosis(Cp) infection on IL-1α maturation and secretion in macrophages and the underlying mechanism.[Methods]The IL-1α expression,maturation and secretion in macrophages infected with Cp (ATCC19410,XH02) and phospholipase D gene (pld) deficient strains (ATCC19410Δpld,XH02Δpld) were evaluated by real-time quantitative PCR,ELISA and Western blotting.Further,the effects of Ca²⁺ chelates (EDTA,EGTA+Mg²⁺ and BAPTA-AM) and calpain inhibitors (calpain inhibitor Ⅲ,calpain inhibitor Ⅳ and EST) on IL-1α maturation and secretion in macrophages infected with Cp were tested.[Results] Cp infection induced increasing of IL-1α mRNA expression and secretion in macrophages,and the IL-1α mRNA expression and secretion in ATCC19410Δpld-infected and XH02Δpld-infected macrophages were significantly decreased compared with those of ATCC19410-infected and XH02-infected macrophages.The intracellular Ca²⁺ concentration and calpain activity were elevated in macrophages after Cp infection,while they were lowered in those infected with pld deficient Cp.Treatment of EDTA,EGTA+Mg²⁺,BAPTA-AM,calpain inhibitor Ⅲ and calpain inhibitor Ⅳ resulted in remarkable decrease of IL-1α expression and secretion in Cp infected macrophages.[Conclusion] Cp infection induced the IL-1α expression,maturation and secretion in macrophages,and phospholipase D was involved in this process.The IL-1α maturation and secretion in macrophages mediated by Cp were related to the increase of intracellular Ca²⁺and the activation of calpain.

Abstract:[Objective] To explore the effect of metal ions against capturing drug resistance gene cassette by integron and the related mechanism.[Methods] An in vivo model of class 1 integron capturing resistance gene cassette was constructed in Escherichia coli.Different concentration of silver ions (0.3,0.9,and 1.5 μg/mL of Ag⁺) and copper ions (5,50,100,150,and 210 μg/mL of Cu²⁺) were used to intervene in the experimental group.The integration frequency of the experimental group and the control group was determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and validated by phenotypic screening.The concentration of metal ions absorbed by bacteria was measured by mass spectrometry,and the molecular mechanism of silver ion against capturing drug resistance gene cassette by bacterial integron was further analyzed by transcriptome sequencing.[Results] The integration frequencies were 9.42×10^‒5(6.49×10^‒5,1.44×10^‒4) in the 0.9 μg/mL silver ion group and 7.29×10^‒5(4.45×10^‒5,9.03×10^‒5) in the 1.5 μg/mL silver ion group,which were lower the 2.59×10^‒4(2.24×10^‒4,3.33×10^‒4) in the control group (P<0.001).In contrast,there was no significant difference between the groups with different concentration of copper ions and the control group.The gene expression level of E.coli before and after silver ion treatment was compared and analyzed by transcriptome sequencing.The differentially expressed genes were related to the Gene Ontology (GO) terms of methylgalactoside transport,maltose transport,phosphoenolpyruvate-glycerone phosphotransferase activity,and glycerone kinase activity,and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of amino sugar and nucleotide sugar metabolism,flagellum assembly,cationic antimicrobial peptide (CAMP) resistance,fructose and mannose metabolism,and phosphotransferase system (PTS).The top 12 hub genes (ptsG,malE,lamB,lacZ,malK,basR,ais,ugd,nagE,metN,malQ and malF) were screened out by protein-protein interaction (PPI) network.[Conclusion] A certain concentration of silver ions inhibited the integration of drug resistance gene cassettes by integron,while the effect of copper ions was not obvious.Therefore,not all metal ions with bactericidal properties had the effect on integration frequency.The mechanism of silver ions against capturing drug resistance gene cassette by bacterial integron was possibly through maltose transport and carbon catabolism.However,the processes of carbon catabolism and maltose transport were complex and deserved further study.At present,there is no related study on bacterial integron transcriptome.This study provides a new way to solve the problem of bacterial drug resistance.

Abstract:[Objective]A murine pneumonia model infected by Pseudomonas aeruginosa was established and treated with a phage cocktail in two ways,and then the therapeutic effect was evaluated.[Methods] We injected 50 μL P.aeruginosa suspension of 2×10⁸ CFU/mL into the trachea after performing the tracheostomy in mice.After half an hour,a phage cocktail preparation was administrated into the infected mice through intranasal and intraperitoneal routes.At the same time,we established two control groups with phosphate-buffered saline in parallel.We then observed the physiological state of the mice and examined the bacteriological,pathological,and inflammatory levels 24 h post infection.[Results]P.aeruginosa was eliminated in the mice administrated with the phage cocktail preparation.The mice treated the phage cocktail showed intact lung tissue structure and slight inflammation compared with the control group.[Conclusion]The phage cocktail preparation can inhibit P.aeruginosa in mice and has a certain therapeutic effect on bacterial infections in the lungs.

Abstract:[Background]Previous studies suggested that the colonization of microorganisms in the host՚s intestinal tract was affected by both host and microbial behaviors,including many factors such as the host's intestinal environment,the origin of microbial strain and species characteristics.It is generally believed that the microbial strains have advantages in colonizing the intestinal tract of the same category of hosts,but there were few reports on the colonization of host intestinal tract at the species and strain levels.[Objective]This study aims to explore the colonization of host guts by different microbial species and strains,and to reveal the key factors affecting the rate of microbial colonization.[Methods]In this study,three strains of Enterococcus xiangfangensis and four strains of Lactobacillus reuteri from Tibetan pigs,ob/ob mice,Macaca fascicularis and fermented food were prepared into a mixed agent to feed germ-free Bama miniature pigs for four weeks,and the colonization of germ-free pig guts by these seven strains were detected with quantitative real-time PCR.[Results]At the species level,E.xiangfangensis and L.reuteri colonized similar number of cells in the germ-free pigs.However,at the strain level,E.xiangfangensis EX-MS and L.reuteri LR-MS that originated from mice colonized more cells than the other five strains,indicating that these mouse-originated strains had an advantage of colonizing germ-free pigs.After the three strains of E.xiangfangensis and four strains of L.reuteri were respectively mixed in equal proportions and cultured for 72 h,E.xiangfangensis EX-MS colonized more cells than EX-MK and EX-PG,while the four strains of L.reuteri colonized similar number of cells.The interaction test between the seven strains demonstrated that EX-MS exerted inhibitory effects on EX-MK and EX-PG,while it had no inhibitory effect on the four strains of L.reuteri.[Conclusion] At the species level,both E.xiangfangensis and L.reuteri had good ability of colonizing the intestinal tract of germ-free pigs.At the strain level,E.xiangfangensis EX-MS and L.reuteri LR-MS that originated from mice had colonization advantages.Moreover,EX-MS gains the colonization advantage in the host intestine by inhibiting the growth of E.xiangfangensis EX-PG and EX-MK respectively originated from Tibetan pigs and M.fascicularis.

Abstract:[Objective] Diguanylate cyclase SiaD regulates the biofilm formation of Pseudomonas aeruginosa.Our previous study about the effect of siaD overexpression on biofilm has revealed that the biofilm yield of a complementary strain is significantly higher than that of the strain overexpressing the wild-type siaD gene.This study aims to explore the reasons for the increase in biofilm production and to study other phenotypes of this strain.[Methods] The mutation sites of siaD were identified by sequencing.The qualitative and quantitative biofilm experiments were carried out to analyze the phenotype of the strain with point mutation.Western blotting was employed to determine the protein level of SiaD^R119M,and GST-pull down assay to measure the binding ability of SiaC to SiaD^R119M in vitro.The fusion expression vector was constructed for the point mutation gene siaD^R119M,and the protein was expressed and purified.The enzyme activity of SiaD^R119M was detected by high performance liquid chromatography (HPLC).Further,we studied the motility of the strain to reveal the relationship between c-di-GMP and bacterial motility.[Results] The sequencing comparison showed that the 119th amino acid was mutated from arginine to methionine (R119M).Compared with that of the wild-type siaD complementary strain,the biofilm yield of siaD^R119A increased,while the biofilm yield of siaD^R119A was lower than that of siaD^R119M;the biofilm yield of siaD^R201A significantly increased and was higher than that of siaD^{R119M R201A}.Western blotting showed no difference in the expression level between SiaD^R119M and wild-type SiaD,and the GST-pull down assay indicated there was a specific interaction between SiaC and SiaD^R119M.The HPLC results showed that the activity of SiaD^R119M decreased.Compared with wild-type siaD complementary strain,siaD^R119M showed weakened motility,and siaD^R201A and siaD^{R119M R201A} had no significant difference in motility.[Conclusion] The mutation of R119M in SiaD increased the biofilm yield,decreased the enzyme activity,and reduced the bacterial motility.This mutation may affect the interaction between SiaD and downstream effector to enhance the signal transduction of downstream effector.The underlying mechanism remains to be explored.

Abstract: Abstract: [Objective] Enterococcus casseliflavus,an opportunistic pathogen,could cause life-threatening sepsis and meningitis.Up to now,little has been known about the phage specifically infecting E.casseliflavus.The isolation,genomic analysis,and characterization of the E.casseliflavus phage can provide a reference for exploring the mechanism of interaction between phage and host and further applying the phage in therapy.[Methods] The phage Ecf_virus_SZ01 was isolated from untreated domestic sewage in Nanshan district,Shenzhen.We employed transmission electron microscopy (TEM) to observe the morphology of Ecf_virus_SZ01 and then studied its biological and genomic characteristics.[Results] Ecf_virus_SZ01 had a non-contractile tail with the length of 150 nm and a head with the diameter of 106 nm,belonging to the Siphoviridae family.The optimal multiplicity of infection (MOI) of the phage was 0.01.The one-step growth curve showed that the phage had an incubation period of about 30 min and the burst size of 50 PFU/cell.Ecf_virus_SZ01 inhibited the growth of host bacteria.Furthermore,the genome of Ecf_virus_SZ01 was 59 409 bp in length,which had the GC content of 43.2% and 102 open reading frames.Blastn comparison showed that this phage had low sequence similarity with other phages deposited in the NCBI database.[Conclusion] In this study,for the first time,we reported a phage that could specifically infect and lyse E.casseliflavus.Both the genomic and phylogenetic analyses indicated the novelty of this phage.

Abstract:[Objective]To investigate the alleviating effect of indole-3-carboxaldehyde (I3A) on the colitis caused by Shigella infection and decipher the underlying mechanism.[Methods]We conducted the in vitro experiment to measure the minimum inhibitory concentration (MIC) and activity of I3A against Shigella by micro broth dilution method.Further,we used a microplate reader to measure the absorbance (A₆₀₀) of the bacterial suspensions with different concentrations of I3A at 600 nm and counted the number of Shigella via the plate colony counting method.In the in vivo experiment,we randomly assigned 18 mice into a control (NC) group,a SF301 group,and a SF301+I3A group.The SF301+I3A group was administrated with 200µL I3A by gavage for 8 consecutive days,and the NC group and SF301 group with 200µL sterile water.The colitis model of Shigella infection was established on the 4th day in the SF301 group and SF301+I3A group.Body weight and disease activity index (DAI) were used to evaluate the conditions of mice.After the above experiments,we euthanized all the mice,collected the middle colon for histopathological detection to observe the inflammation,determined the expression levels of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in the lower colon,and measured the bacterial load in feces and cecum.The safety of I3A application in vivo was monitored in mice for 14 days.[Results] In vitro experiment:the MIC of I3A was 128µg/mL.The bacterial counts and A₆₀₀in the groups treated with different concentrations of I3A were different from those in the control group (P<0.05).In vivo experiment:compared with the SF301 group,the SF301+I3A group showed the body weight and DAI close to those in the NC group and significantly relieved colonic damage.No obvious inflammation was observed in SF301+I3A group,and the bacterial load and the expression levels of inflammatory cytokines were different between SF301+I3A group and SF301 group (P<0.05).I3A had no significant effect on the body weight of mice (P>0.05).[Conclusion] We demonstrate that I3A has a protective effect on the colitis caused by Shigella infection.Specifically,I3A alleviates the colitis by inhibiting Shigella load and reducing colonic damage and inflammation levels.Moreover,I3A shows no toxicity in vivo.

Abstract:[Objective] To analyze the genetic diversity of Vicia faba Rhizobium in arid region of Qinghai,so as to obtain the Rhizobium strain with drought tolerance and matching V.faba varieties and thus promote the application of drought-tolerant Rhizobium of V.faba in arid region of Qinghai.[Methods] With the strain QHCD22 isolated from the arid region of Qinghai as the research subject,species identification and phylogenetic analysis were performed by morphology,physiological and biochemical characteristics,Biolog microbial identification system,16S rRNA gene sequencing and whole genome analysis.Further,polyethylene glycol (PEG)6 000 simulated drought stress,pot experiment and field plot trial were used to evaluate the drought resistance of the strain.[Results] Strain QHCD22 belonged to Rhizobium indicum.With the increased addition of PEG 6 000 to yeast mannitol agar (YMA) broth medium,the strain had an intermediate lethal osmotic potential under medium PEG concentration.But the number of viable strains increased under higher PEG concentration (osmotic potential of−0.6 mPa),and the turbidity of the strain rose from 61.48% to 69.42%,showing a strong drought tolerance.Pot experiment revealed that V.faba inoculated with the strain had higher plant height,GW/DW ratio,nodule number,nodule fresh weight,SPAD values,leaf relative water content (RWC),proline (PRO) content,superoxide dismutase (SOD) activity and root vatality (triphenyltetrazolium chloride,TTC) than that non-inoculated.In addition,under the condition of normal water,the indexes of the inoculated V.faba were higher than those of non-inoculated.The field plot trial indicated that the nitrogenase activity of strain QHCD22 was significantly elevated.Specifically,the nitrogenase activities of Qinghai 13 variety,Qingcan 14 variety and Maya variety ranged from 42.07 to 221.78 C₂H₄ nmol/(g·h),40.60 to 109.78 nmol C₂H₄ nmol/(g·h),and 33.41 to 643.15 C₂H₄ nmol/(g·h),respectively.Rhizobium inoculation promoted the increase of V.faba yield,with Qingcan 14 variety having a significant increase of 32.3%.[Conclusion] Strain QHCD22 belonged to Rhizobium indicum,which had certain drought-tolerant characteristics.The study confirmed that Rhizobium inoculation could improve the drought tolerance of V.faba,especially the drought-sensitive V.faba variety.Therefore the strain had potential application prospects.

Abstract:[Background] Nonstructural protein Nsp2 of coronavirus plays a critical role in the early viral life cycle.[Objective] To explore the host proteins interacting with avian gamma-coronavirus infectious bronchitis virus (IBV) Nsp2 and the interaction of Nsp2 with translation initiation factor 2α(eIF2α).[Methods] We performed the experiment by using the chicken embryo kidney (CEK) cells transfected with recombinant expression vector pCAGGs-Flag-Nsp2 and control vector pCAGGs-Flag.The host cell eIF2α interacting with IBV Nsp2 was obtained by co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS),and then Co-IP and indirect immunofluorescence assay (IFA) were further used to determine the interaction.[Results] A total of 97 candidate proteins were found to interact with Nsp2 through Co-IP and LC-MS/MS,with a high credibility.The key candidate protein eIF2α was further chosen to explore its interaction with Nsp2.The Co-IP and IFA demonstrated that eIF2α and Nsp2 had a direct interrelationship and co-localized in the cytoplasm.Furthermore,the level of eIF2α was upregulated in the overexpression of Nsp2 and IBV-infected CEK cells at the early stage.[Conclusion] In our study,the identification of these candidate proteins interacting with Nsp2 showed that Nsp2 played an important role in IBV infection by manipulating a variety of cellular biological process,and these results provided the basis for further research on the role of Nsp2 in the life cycle of IBV.

Abstract:[Objective] To investigate the effects of arbuscular mycorrhizal fungi (AMF) and dark septate endophyte (DSE) on the yield and quality of Morus alba.[Methods] Pot experiment was conducted,and AMF and DSE were used as bacteria agents for microbial treatment.The effects of AMF and DSE on the growth,yield,and quality of M.alba and the economic potential of microbial agents on the ecological feed crop M.alba were explored by setting up three different microbial combinations (DSE alone,AMF alone,and combined inoculation of DSE+AMF).[Results] Compared with the control check (CK),the three treatments with different bacteria agents significantly promoted the aboveground growth of M.alba and increased the aboveground biomass,with an increase range of 156.4%‒196.6%.Microorganisms with the inoculation of bacteria enhanced the photosynthetic rate of M.alba,and increased the accumulation of nitrogen,phosphorus,and potassium of the leaves,thereby increasing the yield of M.alba.In addition,microorganisms with the inoculation of bacteria increased the crude protein content of leaves and reduced the nutritional value of feed fiber,thus comprehensively improving the quality of M.alba.Microbial treatments significantly increased the leaf-to-stem ratio of M.alba,and increased the proportion of leaves in M.alba,so as to optimize the growth structure.The effect of microbial treatments on the forage quality indexes of M.alba leaves was more significant than that of M.alba stems.Different grades of forage quality were obtained by rationally matching the protein content of leaves and stems,thus improving the overall forage quality of the aboveground part of M.alba.Microorganisms with the inoculation of bacteria greatly improved the forage quality of M.alba,and significantly reduced the content of acid detergent fiber and neutral detergent fiber by 18.4%‒34.6% and 41.0%‒45.4%,respectively.In terms of the M.alba with the inoculation of bacteria,the relative forage value ranged from 220.0 to 241.5,significantly increasing by 0.8 to 1.0 times,and the content of vitamin C,sugar,and alkaloids increased as well.[Conclusion] By the principal component analysis,the order of the comprehensive effect of different bacteria agents on the yield and quality of M.alba was:AMF+DSE>DSE>AMF>CK.DSE,as the bacteria agent for microbial treatment,could optimize M.alba,the economic crops,in the bioremediation of coal mining subsidence areas,and promote the yield and quality of M.alba.

Abstract:[Objective] To screen plant rhizosphere growth-promoting Bacillus velevensis and analyze the biocontrol potential and genome characteristics of the target strain.[Methods]We screened out the growth-promoting strain SF327 through the experiment on pak choi (Brassica campestris sp.chinensis L.) and analysis of plant traits.The activity of strain SF327 against five plant pathogenic fungi and four phytopathogenic bacteria was tested with the filter paper method.Through spray inoculation in paddy fields,the activity of strain SF327 against rice bacterial blight was evaluated.We predicted the secondary metabolites of SF327 using antiSMASH.Moreover,we explored the genetic relationship between SF327 and the plant growth-promoting B.velezensis FZB42 and SQR9,and compared the core genes and secondary metabolite synthesis gene clusters.[Results] The plant growth-promoting SF327 produced auxin indole-3-acetic acid (IAA) and antagonized Magnaporthe oryza,Fusarium oxysporum f.sp.cucumerinum,Phytophthora capsici,Colletotrichum gloeosporioides,and C.acutatum.It had the potential for biocontrol of rice bacterial blight.The genome of SF327 was 4.08 Mb which was composed of a circular chromosome of 4 081 758 base pairs without plasmid and with G+C content of 46.49%,4 033 protein-coding sequences,and 13 gene clusters for secondary metabolites.SF327 strain showed close genetic relationship with strain FZB42 and SQR9,particularly SQR9,and they shared 87% core genes.[Conclusion] B.velezensis SF327 is versatile with a broad antagonistic spectrum,which can be a promising biocontrol agent.