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Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus

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mLife 2024年第1期3卷 119-128页

作者： Pengpeng Zhao Xiaonan Bi Xiaoning Wang Xu Feng Yulong Shen Guanhua Yuan Qunxin She CRISPR and Archaea Biology research Center State Key Laboratory of Microbial Technology and Microbial Technology InstituteShandong UniversityQingdaoChina

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools,which include efficient genome editing,gene silencing,and robust protein expression systems.However,plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid.Although this plasmid coexists with pRN1 in its original host,early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa.islandicus.We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon.We identified a putative target sequence in orf904 encoding a putative replicase on pRN1(target N1).Mutated targets(N1a,N1b,and N1c)were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid inter-ference assay.The results revealed that the original target triggered CRISPR immunity in this archaeon,whereas all three mutated targets did not,indicating that all the designed target mutations evaded host immunity.These mutated targets were then incorporated into orf904 individually,yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed(pN1aSD,pN1bSD,and pN1cSD).Sa.islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors,but pN1cSD lost the capability for replication.These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase.We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination,and this yielded a dual plasmid system for genetic study with this important archaeal model.

关键词： archaeal genetics CRISPR-Cas CRISPR escape mutations dual plasmid vectors Saccharolobus-E.coli shuttle vectors

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Classical taxonomy,molecular phylogeny and genetic analysis of the genus Exitianus Ball,1929(Hemiptera:Cicadellidae:Deltocephalinae)from Egypt

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Zoological Systematics 2024年第2期49卷 143-157页

作者： Rabab M.El-Hady Ahmed F.El-Sayed Survey&Taxonomy department Plant Protection Research InstituteAgriculture Research CentreGizaEgypt microbial Genetics department Biotechnology Research InstituteNational Research CentreGizaEgypt Egypt Center for research and Regenerative Medicine(ECRRM) CairoEgypt

Egyptian species of the leafhopper genus Exitianus Ball,1929,E.capicola(St?l,1855),E.nanus(Distant,1908),and E.pondus Ross,1968 are reviewed.Illustrations,morphological descriptions,and a key for their identification are provided.In this study,we used molecular techniques to confirm morphological identification and detect phylogeny among the three Exitianus species.The partial nucleotide sequences of the amplified products obtained were determined by Macrogen Korea.The nucleotide sequences of 28S rDNA and COX genes of the EGY-ARC-9,EGY-ARC-4,and EGY-ARC-5 were determined by Macrogen Korea,blasted into BLAST at the National Center for Biotechnology Information website(NCBI)and compared with those deposited in the GenBank DNA database.The results represented the homology percentage between the partial sequences of the 28S rRNA and COX genes from each species and related species obtained from GenBank DNA database.The morphological identifications of the three Exitianus species were confirmed by molecular characterization and sequencing of 28S rDNA and COX genes and identified as E.capicola,E.nanus,and E.pondus.Also,their sequences of 28srDNA and COX genes were deposited in GenBank with an accession number(LC670610,LC670607,and OQ196105)for 28srDNA gene and(LC775357,LC775358,and LC775359)for the COX gene.

关键词： Auchenorrhyncha Chaismini taxonomic 28S rDNA phylogenetic

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Occurrence of dark septate endophytes in Phragmites australis in the Baiyang Lake and their resistance to Cd stress

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Pedosphere 2024年第2期34卷 484-496页

作者： Minghui XU Xia LI Qiannan YE Feng GONG Xueli HE School of Life Sciences Hebei UniversityBaoding 071002China Key Laboratory of microbial Diversity research and Application of Hebei Province Hebei UniversityBaoding 071002China

Heavy metal pollution poses a serious hazard to human health,and microbial remediation of heavy metals in soil has been widely studied.A group of ascomycetes classified as dark septate endophytes(DSEs)colonize plant roots and benefit host plants under abiotic stress conditions.In this study,Phragmites australis,a common remediation plant in the Baiyang Lake in North China,was investigated.Soils and roots of P.australis were collected in typical heavy metal-contaminated sites,and the species diversity and community structure of DSEs in P.australis roots were studied.In addition,DSE strains were isolated,cultured,and tested for their tolerance to Cd stress.The results showed that DSEs occurred extensively in P.australis roots,forming typical dark septate hyphae,with a total colonization rate of 19.7%–83.1%.Morphological and internal transcribed spacer sequencing analyses were used to identify 10 species within 9 genera of DSE fungi.Among these fungi,6 strains with considerable resistance to Cd stress were identified.The biomasses of Poaceascoma helicoides,Alternaria doliconidium,and Acrocalymma vagum strains increased as the Cd levels increased.These results can not only help to understand plant-DSE interactions in wetland environments,but also provide a theoretical basis for making full use of DSE fungi to alleviate heavy metal contamination in soil.

关键词： ascomycete colonization rate contaminated site evenness index heavy metal Shannon-Wiener index Simpson index wetland

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Insertion sequence transposition activates antimycobacteriophage immunity through an Isr2-silenced lipid metabolism gene island

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mLife 2024年第1期3卷 87-100页

作者： Yakun Li Yuyun Wei Xiao Guo Xiaohui Li Lining Lu Lihua Hu Zheng-Guo He State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources Guangxi Research Center for Microbial and Enzyme Engineering TechnologyCollege of Life Scienceand TechnologyGuangxi UniversityNanningChina

Insertion sequences(ISs)exist widely in bacterial genomes,but their roles in the evolution of bacterial antiphage defense remain to be clarified.Here,we report that,under the pressure of phage infection,the IS1o96 transposition of Mycobacterium smegmatis into the Isr2 gene can occur at high frequencies,which endows the mutant mycobacterium with a broad-spectrum antiphage ability.Lsr2 functions as a negative regulator and directly silences expression of a gene island composed of 11 lipid metabolism-related genes.The complete or partial loss of the gene island leads to a significant decrease of bacteriophage adsorption to the mycobacterium,thus defending against phage infection.Strikingly,a phage that has evolved mutations in two tail-filament genes can re-escape from the Isr2 inactivation-triggered host defense.This study uncovered a new signaling pathway for activating antimycobacteriophage immunity by Is transposition and provided insight into the natural evolution of bacterial antiphage defense.

关键词： antiphage defense bacteriophage gene island insertion sequence mycobacteria

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Virus-like nanoparticle vaccines for inducing long-lasting immunity against infectious diseases

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National Science Review 2024年第4期11卷 13-15页

作者： Yi-Nan Zhang Sarah Auclair Jiang Zhu department of Integrative Structural and Computational Biology The Scripps Research Institute department of Immunology and Microbiology The Scripps Research Institute

Prophylactic vaccines that can generate long-lasting protective immune responses are needed for many infectious diseases. Efficient vaccination requires vaccine antigens to be retained and presented to B cells in lymp...

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Microstructure and properties of coarse-grained WC-10Co cemented carbides with different carbon contents during heat treatments

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Rare Metals 2024年第4期43卷 1786-1792页

作者： Yuan-Feng Xie Xing-Cheng Xie Zhong-Wu Li Rui-Jun Cao Zhong-Kun Lin Qing Li Chen-Guang Lin Powder Metallurgy and Special Materials research department General Research Institute for Nonferrous Metals

The effects of cryogenic treatment（CT） and tempering-cryogenic treatment（TCT） on the microstructure and properties of coarse-grained WC-10Co cemented carbides with different carbon contents were researched.The binder phase,WC mean grain sizes,W solubility in the binder,relative magnetic s aturation,densities,hardness,wear resistance and second phase precipitation of cemented carbides with different heat treatments were discussed.The results show that there are significant changes of microstructure and properties in the samples with CT and TCT,especially due to the precipitation of metastable nanoparticles WxCoyCzin the binder during the heat treatments of CT and TCT.With the simultaneous combination of microstructure and nanoparticle-reinforced binder,a dramatically improved combination of hardness and wear resistance of the samples after TCT has been achieved.

关键词： Cryogenic treatment Tempering Coarse-grained cemented carbide Carbon content Nanoparticle-reinforced binder

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Combined Treatment with Bojungikgi-tang(Buzhong Yiqi Decoction) and Riluzole Attenuates Cell Death in TDP-43-Expressing Cells

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Chinese Journal of Integrative Medicine 2024年第7期30卷 616-622页

作者： Eun Jin Yang department of KM Science research Korea Institute of Oriental Medicine

Objective: To examine the effect of combined treatment with Bojungikgi-tang(BJIGT, Buzhong Yiqi Decoction) and riluzole(RZ) in transactive response DNA-binding protein 43(TDP-43) stress granule(SG) cells, a amyotrophic lateral sclerosis(ALS) cell line using transcriptomic and molecular techniques. Methods: TDP-43 SG cells were pretreated with BJIGT(100 μg/mL), RZ(50 μmol/L), and combined BJIGT(100 μg/mL)/RZ(50 μmol/L) for 6 h before treatment with lipopolysaccharide(LPS, 200 μmol/L). Cell viability assay was performed to elucidate cell toxicity in TDP-43 SC cells using a cell-counting kit-8(CCK8) assay kit. The expression levels of cell death-related proteins, including Bax, caspase 1, cleaved caspase 3 and DJ1 in TDP-43 SG cells were examined by Western blot analysis. The autophagy-related proteins, including pmTOR/mTOR, LC3b, P62, ATG7 and Bcl-2-associated athanogene 3(Bag3) were investigated using immunofluorescence and immunoblotting assays. Results: Cell viability assay and Western blot analysis showed that combined treatment with BJIGT and RZ suppressed LPS-induced cell death and expression of cell death-related proteins, including Bax, caspase 1, and DJ1(P<0.05 or P<0.01). Immunofluorescence and immunoblotting assays showed that combined treatment with BJIGT and RZ reduced LPS-induced formation of TDP-43 aggregates and regulated autophagy-related protein levels, including p62, light chain 3b, Bag3, and ATG7, in TDP-43-expressing cells(P<0.05 or P<0.01). Conclusion: The combined treatment of BJIGT and RZ might reduce inflammation and regulate autophagy dysfunction in TDP-43-induced ALS.

关键词： Bojungikgi-tang Buzhong Yiqi Decoction transactive response DNA-binding protein 43 autophagy oxidative stress amyotrophic lateral sclerosis

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Gut microbial interactions based on network construction and bacterial pairwise cultivation

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Science China Life Sciences 2024年第8期 1751-1762页

作者： Min-Zhi Jiang Chang Liu Chang Xu He Jiang Yulin Wang Shuang-Jiang Liu State Key Laboratory of microbial Technology Shandong University State Key Laboratory of microbial Resources and Environmental Microbiology Research Center (EMRC)Institute of MicrobiologyChinese Academy of Sciences University of Chinese Academy of Sciences

Association networks are widely applied for the prediction of bacterial interactions in studies of human gut microbiomes.However,the experimental validation of the predicted interactions is challenging due to the complexity of gut microbiomes and the limited number of cultivated bacteria.In this study,we addressed this challenge by integrating in vitro time series network(TSN) associations and cocultivation of TSN taxon pairs.Fecal samples were collected and used for cultivation and enrichment of gut microbiome on YCFA agar plates for 13 days.Enriched cells were harvested for DNA extraction and metagenomic sequencing.A total of 198 metagenome-assembled genomes(MAGs) were recovered.Temporal dynamics of bacteria growing on the YCFA agar were used to infer microbial association networks.To experimentally validate the interactions of taxon pairs in networks,we selected 24 and 19 bacterial strains from this study and from the previously established human gut microbial biobank,respectively,for pairwise co-cultures.The co-culture experiments revealed that most of the interactions between taxa in networks were identified as neutralism(51.67%),followed by commensalism(21.67%),amensalism(18.33%),competition(5%) and exploitation(3.33%).Genome-centric analysis further revealed that the commensal gut bacteria(helpers and beneficiaries) might interact with each other via the exchanges of amino acids with high biosynthetic costs,short-chain fatty acids,and/or vitamins.We also validated 12 beneficiaries by adding 16 additives into the basic YCFA medium and found that the growth of 66.7% of these strains was significantly promoted.This approach provides new insights into the gut microbiome complexity and microbial interactions in association networks.Our work highlights that the positive relationships in gut microbial communities tend to be overestimated,and that amino acids,short-chain fatty acids,and vitamins are contributed to the positive relationships.

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Cultivating the uncultured:Harnessing the“sandwich agar plate”approach to isolate heme-dependent bacteria from marine sediment

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mLife 2024年第1期3卷 143-155页

作者： Jing Zhang Qi-Yun Liang Da-Shuai Mu Fengbai Lian Ya Gong Mengqi Ye Guan-Jun Chen Yuqi Ye Zong-Jun Du State Key Laboratory of microbial Techlogy Insttute of Micrbial TchnoogyShandong UniversityQingdaoChina Marine College Shandong UniverstyWeihaiChina Shandong University-Weihai research Institute of Industrial Technology WeihaiChina

In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival.In this study,we designed a simple coculture technique named the“sandwich agar plate method,”which maintains microbial interactions throughout the isolation and pure culture processes.The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group.Many uncultured species displayed commensal lifestyles.Further study found that heme was the growth-promoting factor of some marine commensal bacteria.Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa.Moreover,our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably.These findings highlight that cocultivation based on the“sandwich agar plate method”could be developed and used to isolate more uncultured bacteria.

关键词： cultivation growth factors heme sandwich agar plate uncultured bacteria

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Mycorrhizal fungi mitigate nitrogen losses of an experimental grassland by facilitating plant uptake and soil microbial immobilization

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Pedosphere 2024年第2期34卷 399-410页

作者： Yangyang JIA Marcel G.A.VAN DER HEIJDEN Alain Y.VALZANO-HELD Markus JOCHER Florian WALDER Postdoctoral Mobile Station of Ecology College of Ecology and EnvironmentXinjiang UniversityÜrümqi 830046China research Group Plant-Soil Interactions AgroscopeZürich 8046Switzerland research Group Climate and Agriculture AgroscopeZürich 8046Switzerland department of Plant and microbial Biology University of ZürichZürich 8008Switzerland research Group Soil Quality and Soil Use AgroscopeZürich 8046Switzerland

Nitrogen(N)is one of the most limited nutrients of terrestrial ecosystems,whose losses are prevented in tightly coupled cycles in finely tuned systems.Global change-induced N enrichment through atmospheric deposition and application of vast amounts of fertilizer are now challenging the terrestrial N cycle.Arbuscular mycorrhizal fungi(AMF)are known drivers of plant-soil nutrient fluxes,but a comprehensive assessment of AMF involvement in N cycling under global change is still lacking.Here,we simulated N enrichment by fertilization(low/high)in experimental grassland microcosms under greenhouse conditions in the presence or absence of AMF and continuously monitored different N pathways over nine months.We found that high N enrichment by fertilization decreased the relative abundance of legumes and the plant species dominating the plant community changed from grasses to forbs in the presence of AMF,based on aboveground biomass.The presence of AMF always maintained plant N:phosphorus(P)ratios between 14 and 16,no matter how the soil N availability changed.Shifts in plant N:P ratios due to the increased plant N and P uptake might thus be a primary pathway of AMF altering plant community composition.Furthermore,we constructed a comprehensive picture of AMF’s role in N cycling,highlighting that AMF reduced N losses primarily by mitigating N leaching,while N_(2)O emissions played a marginal role.Arbuscular mycorrhizal fungi reduced N_(2)O emissions directly through the promotion of N_(2)O-consuming denitrifiers.The underlying mechanism for reducing N leaching is mainly the AMF-mediated improved nutrient uptake and AMF-associated microbial immobilization.Our results indicate that synergies between AMF and other soil microorganisms cannot be ignored in N cycling and that the integral role of AMF in N cycling terrestrial ecosystems can buffer the upcoming global changes.

关键词： N cycling N enrichment N uptake N:P ratio plant community structure symbiotic soil fungi

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