The objective of this study is to quantitatively reveal the main genetic carrier of antibiotic resistance genes(ARGs)for blocking their environmental dissemination.The distribution of ARGs in chromosomes,plasmids,and ...The objective of this study is to quantitatively reveal the main genetic carrier of antibiotic resistance genes(ARGs)for blocking their environmental dissemination.The distribution of ARGs in chromosomes,plasmids,and phages for understanding their respective contributions to the development of antimicrobial resistance in aerobic biofilm consortium under increasing stresses of oxytetracycline,streptomycin,and tigecyclinewere revealed based on metagenomics analysis.Results showed that the plasmids harbored 49.2%-83.9%of resistomes,which was higher(p<0.001)than chromosomes(2.0%-35.6%),and no ARGs were detected in phage contigs under the strict alignment standard of over 80%identity used in this study.Plasmids and chromosomes tended to encode different types of ARGs,whose abundances all increased with the hike of antibiotic concentrations,and the variety of ARGs encoded by plasmids(14 types and 64 subtypes)was higher than that(11 types and 27 subtypes)of chromosomes.The dosing of the three antibiotics facilitated the transposition and recombination of ARGs on plasmids,mediated by transposable and integrable transfer elements,which increased the co-occurrence of associated and unassociated ARGs.The results quantitatively proved that plasmids dominate the proliferation of ARGs in aerobic biofilm driven by antibiotic selection,which should be a key target for blocking ARG dissemination.展开更多
With the increasing accumulation of plastic pollutants in various environments,research on microorganisms(including bacteria,fungi,and algae)with plastic degradation capabilities has gained significant attention.Howev...With the increasing accumulation of plastic pollutants in various environments,research on microorganisms(including bacteria,fungi,and algae)with plastic degradation capabilities has gained significant attention.However,only a limited number of microbial plastic-degrading enzymes have been identified to date.This highlights that the degradation mechanisms employed by many plastic-degrading microorganisms,particularly filamentous fungi,remain insufficiently explored.In this study,we utilized a versatile fungal plasmid(pCT74)to express green fluorescent protein(GFP)in a marine-derived fungus Alternaria alternata strain FB1 with plastic degradation capabilities.Upon evaluating the degradation effect of polyester-type polyurethane(PU)film,we observed that different transformants exhibited three kinds of activities(the same,reduced,or enhanced degradation capability)compared to the FB1 wild-type strain.Further analysis of the plasmid fragment insertion sites in different transformants revealed that pCT74 integrates randomly into the genome of the host fungus.Notably,a direct correlation was found between the plasmid insertion site and the degradation capability of the corresponding transformant.Our findings not only redefine the potential applications of plasmid pCT74 in filamentous fungi but also show a novel research approach to identifying key enzymes involved in plastic degradation by fungi.展开更多
Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especiall...Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.展开更多
QuickChange mutagenesis is the method of choice for site-directed mutagenesis （SDM） of target sequences in a plasmid. It can be applied successfully to small plasmids （up to 10 kb）. However, this method cannot eff...QuickChange mutagenesis is the method of choice for site-directed mutagenesis （SDM） of target sequences in a plasmid. It can be applied successfully to small plasmids （up to 10 kb）. However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography （HPLC） purified primers, we were able to achieve SDM in big plasmids （up to 16 kb） involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction （PCR） cycles and 0.5μI of polymerase （instead of 18 PCR cycles and 1.0 μI of enzyme in the standard protocol） were sufficient for the reaction.展开更多
To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression...To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression vector pVAX1, These four plasmids were identified by restriction enzyme digestion and gene sequencing. The plasmid encoding the N protein was selected to determine the expression effect of these plasmids in NA cells. The results showed that the helper plasmids for a reverse genetics system of rabies street virus strain HN10 had been successfully constructed.展开更多
Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable marke...Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.展开更多
After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the ...After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the effect of excessive CdCl 2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl 2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl 2 did not inhibit the replication and amp + genes expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl 2 treatment for 24 hours might cause the sequences change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl 2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl 2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.展开更多
Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this meth...Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.展开更多
Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of tran...Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of transformed Escherichia coli (E. coli) in liquid medium, brief centrifugation for precipitation of bacterial pellets, and subsequent lysis of the pellets. This process is time-consuming and laborious, especially when the sample number is high. Here, we describe a more convenient method for MiniPrep analysis that utilizes solid medium-based cultivation of bacteria.展开更多
Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished f...Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.展开更多
HighlightsA novel conjugative plasmid pHJ90-cfr carrying the multiresistance gene cfr was characterized in Proteus vulgaris.A new IS5-family member,ISPmi4,was identified for the first time.Both plasmids and ICEs were ...HighlightsA novel conjugative plasmid pHJ90-cfr carrying the multiresistance gene cfr was characterized in Proteus vulgaris.A new IS5-family member,ISPmi4,was identified for the first time.Both plasmids and ICEs were vital mobile genetic elements for horizontal transmission of cfr gene in Proteus species.展开更多
As an emerging environmental contaminant,antibiotic resistance genes(ARGs)in tap water have attracted great attention.Although studies have provided ARG profiles in tap water,research on their abundance levels,composi...As an emerging environmental contaminant,antibiotic resistance genes(ARGs)in tap water have attracted great attention.Although studies have provided ARG profiles in tap water,research on their abundance levels,composition characteristics,and potential threat is still insufficient.Here,9 household tap water samples were collected from the Guangdong-Hong Kong-Macao Greater Bay Area(GBA)in China.Additionally,75 sets of environmental sample data(9 types)were downloaded from the public database.Metagenomics was then performed to explore the differences in the abundance and composition of ARGs.221 ARG subtypes consisting of 17 types were detected in tap water.Although the ARG abundance in tap water was not significantly different from that found in drinking water plants and reservoirs,their composition varied.In tap water samples,the three most abundant classes of resistance genes were multidrug,fosfomycin and MLS(macrolide-lincosamidestreptogramin)ARGs,and their corresponding subtypes ompR,fosX and macB were also the most abundant ARG subtypes.Regarding the potential mobility,vanS had the highest abundance on plasmids and viruses,but the absence of key genes rendered resistance to vancomycin ineffective.Generally,the majority of ARGs present in tap water were those that have not been assessed and are currently not listed as high-threat level ARG families based on the World Health Organization Guideline.Although the current potential threat to human health posed by ARGs in tap water is limited,with persistent transfer and accumulation,especially in pathogens,the potential danger to human health posed by ARGs should not be ignored.展开更多
The escalating global dissemination of plasmid-mediated antibiotic resistance poses a formidable threat to global health.Conjugation stands as a pivotal mechanism for horizontal gene transfer among bacterial populatio...The escalating global dissemination of plasmid-mediated antibiotic resistance poses a formidable threat to global health.Conjugation stands as a pivotal mechanism for horizontal gene transfer among bacterial populations,facilitating the spread of antibiotic resistance genes(ARGs).Microelectrolysis has garnered attention as an efficacious strategy for mitigating antibiotic concentrations in wastewater,yet its potential impact on ARG horizontal transfer remain largely unexplored.This comprehensive investigation unveils that microelectrolysis not only influences but significantly accelerates the conjugative transfer of ARG-harboring plasmids.Remarkably,this phenomenon is corroborated at the microbial community scale,underscoring its ecological relevance.Alarmingly,the study highlights the vulnerability of intestinalmicroorganisms to acquire antibiotic resistance under electrolytic stimulation,posing heightened risks to both animal and human health.Delving deeper,the study elucidates the underlyingmechanisms responsible for this enhanced conjugative transfer.It reveals that microelectrolysis augments the abundance of mating-competent cells,triggers the generation of reactive oxygen species,inflicts modest membrane damage,and upregulates the expression of genes critical for conjugation.These findings collectively contribute to a more profound comprehension of the environmental dissemination dynamics and associated public health implications of ARGs in the context of wastewater treatment employing microelectrolytic technologies.展开更多
Herpes simplex virus thymidine kinase(HSVtk)gene therapy is a promising strategy for glioblastoma therapy.However,delivery of plasmid DNA(pDNA)encoding HSVtk into the brain by systemic administration is a challenge si...Herpes simplex virus thymidine kinase(HSVtk)gene therapy is a promising strategy for glioblastoma therapy.However,delivery of plasmid DNA(pDNA)encoding HSVtk into the brain by systemic administration is a challenge since pDNA can hardly penetrate the bloodbrain barrier.In this study,an exosome-membrane(EM)and polymer-based hybrid complex was developed for systemic delivery of pDNA into the brain.Histidine/arginine-linked polyamidoamine(PHR)was used as a carrier.PHR binds to pDNA by electrostatic interaction.The pDNA/PHR complex was mixed with EM and subjected to extrusion to produce pDNA/PHR-EM hybrid complex.For glioblastoma targeting,T7 peptide was attached to the pDNA/PHR-EM complex.Both pDNA/PHR-EM and T7-decorated pDNA/PHR-EM(pDNA/PHREM-T7)had a surface charge of–5 mV and a size of 280 nm.Transfection assays indicated that pDNA/PHR-EM-T7 enhanced the transfection to C6 cells compared with pDNA/PHREM.Intravenous administration of pHSVtk/PHR-EM-T7 showed that pHSVtk/PHR-EM and pHSVtk/PHR-EM-T7 delivered pHSVtk more efficiently than pHSVtk/lipofectamine and pHSVtk/PHR into glioblastoma in vivo.pHSVtk/PHR-EM-T7 had higher delivery efficiency than pHSVtk/PHR-EM.As a result,the HSVtk expression and apoptosis levels in the tumors of the pHSVtk/PHR-EM-T7 group were higher than those of the other control groups.Therefore,the pDNA/PHR-EM-T7 hybrid complex is a useful carrier for systemic delivery of pHSVtk to glioblastoma.展开更多
Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication syste...Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication system constructed based on B.thuringiensis prophage GIL16,can achieve stable and high levels of gene expression in the absence of external selection pressure,facilitating development of B.thuringiensis chassis cells.However,the regulatory elements of gene expression and protein secretion suitable for the B.thuringiensis expression system are still lacking.Therefore,the development and optimization of different genetic tools are required.We constructed a promoter library containing 107 different-strength promoters(covering persistently high/intermediate/low level)by transcriptomic analysis of the cell at different growth stages and a signal peptide library(59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B.thuringiensis)to enrich the genetic toolbox using alpha-lactalbumin(α-LA)as the characterization product.Then,a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements,resulting in an improved α-LA-producing B.thuringiensis.Finally,the maximum copy number of linear plasmids was 9.3 times higher than that of the original.The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor,which was comparable to the highest yield reported in Komagataella phaffii.We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.展开更多
The inappropriate use of cephalosporins lead to the occurrence and global spread of bacteria resistant to these antimicrobials.In this study,we isolated four Escherichia albertii strains from broilers in eastern China...The inappropriate use of cephalosporins lead to the occurrence and global spread of bacteria resistant to these antimicrobials.In this study,we isolated four Escherichia albertii strains from broilers in eastern China.The antimicrobial susceptibility and genomic characterization of these E.albertii isolates were determined.Our results revealed that these four E.albertii isolates exhibited resistance to tetracyclines,chloramphenicol,β-lactams,aminoglycosides,polymyxin B,sulfonamides,quinolones,and other antimicrobials.Among them,EA04 isolate was multidrug resistant and harbored extended-spectrumβ-lactamases(ESBL)genes blaCTX-Mand blaTEM.Whole genome sequencing and core-genome multilocus sequence typing(cgMLST)based on all ST4638 E.albertii for EA04 inferred highly probable epidemiological links between selected human isolates.Additionally,the ESBL genes blaTEM-141and blaCTX-M-55were coexistent in an approximately 75 kb Inc FII plasmid pEA04.2 in EA04.Comparative analysis indicated that genes blaTEM-141and blaCTX-M-55were located in IS15-blaCTX-M-55-wbu C-blaTEM-141-IS26 region,which similar structures were identified in various bacteria.Furthermore,the plasmid pEA04.2 could be transferable to E.coli EC600 and lead to the resistance to third-generation cephalosporins.These results suggested that chicken potentially serve as a reservoir for multidrug resistant E.albertii,which increases the risk of horizontal transfer of antimicrobial resistance between humans,animals and environment.展开更多
Microneedle technology is valuable in wound treatment.Current studies focus on optimizing the function of microneedles and screening for effective encapsulated actives.Herein,we develop innovative MXene hydrogel micro...Microneedle technology is valuable in wound treatment.Current studies focus on optimizing the function of microneedles and screening for effective encapsulated actives.Herein,we develop innovative MXene hydrogel microneedles with nitric oxide(NO)and hypoxia-inducible factor-1α(HIF-1α)plasmid controllable release for diabetic wound treatment.These microneedles consist of gelatin coupled with tert-butyl nitrite(Gel-SNO)polymers obtained by conjugating the-SNO group on the gelatin side chain,therefore,NO can be generated and released under near-infra red(NIR)light irradiation owing to the thermal effect.Simultaneously,by harnessing the enhanced photothermal conversion efficiency of the MXene additive,the microneedle patch can quickly dissolve and liberate the enclosed HIF-1αplasmid nanoparticles into the dermis when exposed to NIR radiation.The released NO effectively reduced the inflammatory response and released HIF-1αplasmid induced neovascularization.Thus,in vivo experiments showed that these microneedles could accelerate wound closure by alleviating inflammation,and promoting re-epithelialization and angiogenesis.These results indicated the potential value of MXene hydrogel microneedles in wound healing and other related biomedical fields.展开更多
Protein Kinase G(PKG)is an important intracellular signal transduction enzyme,and its activity is modulated by cyclic guanosine monophosphate(cGMP).PKG plays a pivotal role in various significant physiological process...Protein Kinase G(PKG)is an important intracellular signal transduction enzyme,and its activity is modulated by cyclic guanosine monophosphate(cGMP).PKG plays a pivotal role in various significant physiological processes,including vascular smooth muscle relaxation,myocardial cell function regulation,neuron growth,and synaptic plasticity,et al.In recent years,the role of PKG in diseases has gradually attracted attention,and the abnormalities in its signaling pathway are closely related to the occurrence and development of cardiovascular and neurological diseases.Although PKG has been widely studied,its complex functions in different physiological systems and potential innovative applications still need to be further explored.This article reviews the purification techniques for PKG,discusses the advantages and disadvantages of different extraction methods,summarizes the structure and activation mechanism of each domain of PKG,and analyzes the physiological functions of PKG in organisms,especially the well-established roles in the cardiovascular system,nervous system,and endocrine system.The emerging therapeutic applications of PKG are also reviewed.In addition,the challenges of this field are proposed at the end.展开更多
[Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 p...[Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 pathogenic Escherichia coli in musk deer were extracted by the Lysis Triton method, and then identified by single enzyme digestion with three endonucleases of Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ. [Result] The yield rate of plasmids was 91.6%, and 24 pathogenic Escherichia coli in musk deer had the identical or similar plasmid profiles. [Conclusion] Plasmid DNA analysis offers scientific basis for molecular epidemiology of pathogenic Escherichia coli in musk deer in Sichuan Institute of Musk Deer Breeding.展开更多
基金supported by the National Natural Science Foundation of China(Nos.52091545 and 51978645).
文摘The objective of this study is to quantitatively reveal the main genetic carrier of antibiotic resistance genes(ARGs)for blocking their environmental dissemination.The distribution of ARGs in chromosomes,plasmids,and phages for understanding their respective contributions to the development of antimicrobial resistance in aerobic biofilm consortium under increasing stresses of oxytetracycline,streptomycin,and tigecyclinewere revealed based on metagenomics analysis.Results showed that the plasmids harbored 49.2%-83.9%of resistomes,which was higher(p<0.001)than chromosomes(2.0%-35.6%),and no ARGs were detected in phage contigs under the strict alignment standard of over 80%identity used in this study.Plasmids and chromosomes tended to encode different types of ARGs,whose abundances all increased with the hike of antibiotic concentrations,and the variety of ARGs encoded by plasmids(14 types and 64 subtypes)was higher than that(11 types and 27 subtypes)of chromosomes.The dosing of the three antibiotics facilitated the transposition and recombination of ARGs on plasmids,mediated by transposable and integrable transfer elements,which increased the co-occurrence of associated and unassociated ARGs.The results quantitatively proved that plasmids dominate the proliferation of ARGs in aerobic biofilm driven by antibiotic selection,which should be a key target for blocking ARG dissemination.
基金Supported by the Science and Technology Innovation Project of Laoshan Laboratory(Nos.2022QNLM030004-3,LSKJ202203103)the NSFC Innovative Group Grant(No.42221005)+5 种基金the Key Collaborative Research Program of the Alliance of International Science Organizations(No.ANSO-CR-KP-2022-08)the Shandong Provincial Natural Science Foundation(No.ZR2021ZD28)the Major Research Plan of the National Natural Science Foundation(No.92351301)the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDA22050301)the Taishan Scholars Program(No.tstp20230637)the Qingdao Natural Science Foundation(No.23-2-1-182-zyyd-jch)。
文摘With the increasing accumulation of plastic pollutants in various environments,research on microorganisms(including bacteria,fungi,and algae)with plastic degradation capabilities has gained significant attention.However,only a limited number of microbial plastic-degrading enzymes have been identified to date.This highlights that the degradation mechanisms employed by many plastic-degrading microorganisms,particularly filamentous fungi,remain insufficiently explored.In this study,we utilized a versatile fungal plasmid(pCT74)to express green fluorescent protein(GFP)in a marine-derived fungus Alternaria alternata strain FB1 with plastic degradation capabilities.Upon evaluating the degradation effect of polyester-type polyurethane(PU)film,we observed that different transformants exhibited three kinds of activities(the same,reduced,or enhanced degradation capability)compared to the FB1 wild-type strain.Further analysis of the plasmid fragment insertion sites in different transformants revealed that pCT74 integrates randomly into the genome of the host fungus.Notably,a direct correlation was found between the plasmid insertion site and the degradation capability of the corresponding transformant.Our findings not only redefine the potential applications of plasmid pCT74 in filamentous fungi but also show a novel research approach to identifying key enzymes involved in plastic degradation by fungi.
基金the Wellcome Trust,BBSRC,and the National Natural Science Foundation of China(81802065,102908/Z/13/Z).
文摘Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.
文摘QuickChange mutagenesis is the method of choice for site-directed mutagenesis （SDM） of target sequences in a plasmid. It can be applied successfully to small plasmids （up to 10 kb）. However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography （HPLC） purified primers, we were able to achieve SDM in big plasmids （up to 16 kb） involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction （PCR） cycles and 0.5μI of polymerase （instead of 18 PCR cycles and 1.0 μI of enzyme in the standard protocol） were sufficient for the reaction.
基金National High Technology Research and Development Program of China (2006AA02Z110, 2007AA02Z402)Major Program of the National Natural Science Foundation of China (30630049)
文摘To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression vector pVAX1, These four plasmids were identified by restriction enzyme digestion and gene sequencing. The plasmid encoding the N protein was selected to determine the expression effect of these plasmids in NA cells. The results showed that the helper plasmids for a reverse genetics system of rabies street virus strain HN10 had been successfully constructed.
基金Supported by the National Basic Research Program of China(973Program)(No.2009CB118703)
文摘Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.
文摘After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the effect of excessive CdCl 2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl 2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl 2 did not inhibit the replication and amp + genes expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl 2 treatment for 24 hours might cause the sequences change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl 2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl 2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.
文摘Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.
文摘Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of transformed Escherichia coli (E. coli) in liquid medium, brief centrifugation for precipitation of bacterial pellets, and subsequent lysis of the pellets. This process is time-consuming and laborious, especially when the sample number is high. Here, we describe a more convenient method for MiniPrep analysis that utilizes solid medium-based cultivation of bacteria.
文摘Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.
基金supported by the National Key Research and Development Program of China(2022YFF0710505)the Central Public-interest Scientific Institution Basal Research Fund,China(1610302022001)。
文摘HighlightsA novel conjugative plasmid pHJ90-cfr carrying the multiresistance gene cfr was characterized in Proteus vulgaris.A new IS5-family member,ISPmi4,was identified for the first time.Both plasmids and ICEs were vital mobile genetic elements for horizontal transmission of cfr gene in Proteus species.
基金supported by the National Key R&D Program of China(No.2022YFE0103200)the Hubei Provincial Natural Science Foundation of China(No.2021CFB016)the National Natural Science Foundation of China(No.52100217).
文摘As an emerging environmental contaminant,antibiotic resistance genes(ARGs)in tap water have attracted great attention.Although studies have provided ARG profiles in tap water,research on their abundance levels,composition characteristics,and potential threat is still insufficient.Here,9 household tap water samples were collected from the Guangdong-Hong Kong-Macao Greater Bay Area(GBA)in China.Additionally,75 sets of environmental sample data(9 types)were downloaded from the public database.Metagenomics was then performed to explore the differences in the abundance and composition of ARGs.221 ARG subtypes consisting of 17 types were detected in tap water.Although the ARG abundance in tap water was not significantly different from that found in drinking water plants and reservoirs,their composition varied.In tap water samples,the three most abundant classes of resistance genes were multidrug,fosfomycin and MLS(macrolide-lincosamidestreptogramin)ARGs,and their corresponding subtypes ompR,fosX and macB were also the most abundant ARG subtypes.Regarding the potential mobility,vanS had the highest abundance on plasmids and viruses,but the absence of key genes rendered resistance to vancomycin ineffective.Generally,the majority of ARGs present in tap water were those that have not been assessed and are currently not listed as high-threat level ARG families based on the World Health Organization Guideline.Although the current potential threat to human health posed by ARGs in tap water is limited,with persistent transfer and accumulation,especially in pathogens,the potential danger to human health posed by ARGs should not be ignored.
基金supported by Jiangsu Agriculture Science and Technology Innovation Fund(No.CX(22)3001)。
文摘The escalating global dissemination of plasmid-mediated antibiotic resistance poses a formidable threat to global health.Conjugation stands as a pivotal mechanism for horizontal gene transfer among bacterial populations,facilitating the spread of antibiotic resistance genes(ARGs).Microelectrolysis has garnered attention as an efficacious strategy for mitigating antibiotic concentrations in wastewater,yet its potential impact on ARG horizontal transfer remain largely unexplored.This comprehensive investigation unveils that microelectrolysis not only influences but significantly accelerates the conjugative transfer of ARG-harboring plasmids.Remarkably,this phenomenon is corroborated at the microbial community scale,underscoring its ecological relevance.Alarmingly,the study highlights the vulnerability of intestinalmicroorganisms to acquire antibiotic resistance under electrolytic stimulation,posing heightened risks to both animal and human health.Delving deeper,the study elucidates the underlyingmechanisms responsible for this enhanced conjugative transfer.It reveals that microelectrolysis augments the abundance of mating-competent cells,triggers the generation of reactive oxygen species,inflicts modest membrane damage,and upregulates the expression of genes critical for conjugation.These findings collectively contribute to a more profound comprehension of the environmental dissemination dynamics and associated public health implications of ARGs in the context of wastewater treatment employing microelectrolytic technologies.
基金supported by the Individual Basic Science&Engineering Research Program(NRF-2022R1A2B5B01001920)through the National Research Foundation,funded by the Ministry of Science and ICT in Korea.
文摘Herpes simplex virus thymidine kinase(HSVtk)gene therapy is a promising strategy for glioblastoma therapy.However,delivery of plasmid DNA(pDNA)encoding HSVtk into the brain by systemic administration is a challenge since pDNA can hardly penetrate the bloodbrain barrier.In this study,an exosome-membrane(EM)and polymer-based hybrid complex was developed for systemic delivery of pDNA into the brain.Histidine/arginine-linked polyamidoamine(PHR)was used as a carrier.PHR binds to pDNA by electrostatic interaction.The pDNA/PHR complex was mixed with EM and subjected to extrusion to produce pDNA/PHR-EM hybrid complex.For glioblastoma targeting,T7 peptide was attached to the pDNA/PHR-EM complex.Both pDNA/PHR-EM and T7-decorated pDNA/PHR-EM(pDNA/PHREM-T7)had a surface charge of–5 mV and a size of 280 nm.Transfection assays indicated that pDNA/PHR-EM-T7 enhanced the transfection to C6 cells compared with pDNA/PHREM.Intravenous administration of pHSVtk/PHR-EM-T7 showed that pHSVtk/PHR-EM and pHSVtk/PHR-EM-T7 delivered pHSVtk more efficiently than pHSVtk/lipofectamine and pHSVtk/PHR into glioblastoma in vivo.pHSVtk/PHR-EM-T7 had higher delivery efficiency than pHSVtk/PHR-EM.As a result,the HSVtk expression and apoptosis levels in the tumors of the pHSVtk/PHR-EM-T7 group were higher than those of the other control groups.Therefore,the pDNA/PHR-EM-T7 hybrid complex is a useful carrier for systemic delivery of pHSVtk to glioblastoma.
基金financially supported by the National Science Fund for Excellent Young Scholars(32222069)National Natural Science Foundation of China(32172349)+1 种基金Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005)National Key Research and Development Program of China(2020YFA0908300).
文摘Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication system constructed based on B.thuringiensis prophage GIL16,can achieve stable and high levels of gene expression in the absence of external selection pressure,facilitating development of B.thuringiensis chassis cells.However,the regulatory elements of gene expression and protein secretion suitable for the B.thuringiensis expression system are still lacking.Therefore,the development and optimization of different genetic tools are required.We constructed a promoter library containing 107 different-strength promoters(covering persistently high/intermediate/low level)by transcriptomic analysis of the cell at different growth stages and a signal peptide library(59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B.thuringiensis)to enrich the genetic toolbox using alpha-lactalbumin(α-LA)as the characterization product.Then,a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements,resulting in an improved α-LA-producing B.thuringiensis.Finally,the maximum copy number of linear plasmids was 9.3 times higher than that of the original.The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor,which was comparable to the highest yield reported in Komagataella phaffii.We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.
基金supported by the National Key Research and Development Program of China(2021YFD1800402)the National Natural Science Foundation of China(32172856,31972654 and 32302881)+1 种基金the Natural Science Foundation of Shanghai,China(22ZR1476100 and 23ZR1476600)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences(SHVRI-ASTIP-2014-8)。
文摘The inappropriate use of cephalosporins lead to the occurrence and global spread of bacteria resistant to these antimicrobials.In this study,we isolated four Escherichia albertii strains from broilers in eastern China.The antimicrobial susceptibility and genomic characterization of these E.albertii isolates were determined.Our results revealed that these four E.albertii isolates exhibited resistance to tetracyclines,chloramphenicol,β-lactams,aminoglycosides,polymyxin B,sulfonamides,quinolones,and other antimicrobials.Among them,EA04 isolate was multidrug resistant and harbored extended-spectrumβ-lactamases(ESBL)genes blaCTX-Mand blaTEM.Whole genome sequencing and core-genome multilocus sequence typing(cgMLST)based on all ST4638 E.albertii for EA04 inferred highly probable epidemiological links between selected human isolates.Additionally,the ESBL genes blaTEM-141and blaCTX-M-55were coexistent in an approximately 75 kb Inc FII plasmid pEA04.2 in EA04.Comparative analysis indicated that genes blaTEM-141and blaCTX-M-55were located in IS15-blaCTX-M-55-wbu C-blaTEM-141-IS26 region,which similar structures were identified in various bacteria.Furthermore,the plasmid pEA04.2 could be transferable to E.coli EC600 and lead to the resistance to third-generation cephalosporins.These results suggested that chicken potentially serve as a reservoir for multidrug resistant E.albertii,which increases the risk of horizontal transfer of antimicrobial resistance between humans,animals and environment.
基金supported by the National Key Research and Development Program of China(2022YFA1105300)the Key Research&Developement(R&D)Program of Jiangsu Province(BE2022853)+2 种基金the Joint Fund of Henan Province Science and Technology R&D Program(225200810021)the Clinical Trials from Nanjing Drum Tower Hospital(2022-LCYJ-ZD-01)theJiangsu Funding Program for Excellent Postdoctoral Talent(2024ZB003)。
文摘Microneedle technology is valuable in wound treatment.Current studies focus on optimizing the function of microneedles and screening for effective encapsulated actives.Herein,we develop innovative MXene hydrogel microneedles with nitric oxide(NO)and hypoxia-inducible factor-1α(HIF-1α)plasmid controllable release for diabetic wound treatment.These microneedles consist of gelatin coupled with tert-butyl nitrite(Gel-SNO)polymers obtained by conjugating the-SNO group on the gelatin side chain,therefore,NO can be generated and released under near-infra red(NIR)light irradiation owing to the thermal effect.Simultaneously,by harnessing the enhanced photothermal conversion efficiency of the MXene additive,the microneedle patch can quickly dissolve and liberate the enclosed HIF-1αplasmid nanoparticles into the dermis when exposed to NIR radiation.The released NO effectively reduced the inflammatory response and released HIF-1αplasmid induced neovascularization.Thus,in vivo experiments showed that these microneedles could accelerate wound closure by alleviating inflammation,and promoting re-epithelialization and angiogenesis.These results indicated the potential value of MXene hydrogel microneedles in wound healing and other related biomedical fields.
基金supported by the National Natural Science Foundation of China(Nos.22374033,22174031,22407037)the Natural Science Foundation of Heilongjiang Province(No.ZD2022B001).
文摘Protein Kinase G(PKG)is an important intracellular signal transduction enzyme,and its activity is modulated by cyclic guanosine monophosphate(cGMP).PKG plays a pivotal role in various significant physiological processes,including vascular smooth muscle relaxation,myocardial cell function regulation,neuron growth,and synaptic plasticity,et al.In recent years,the role of PKG in diseases has gradually attracted attention,and the abnormalities in its signaling pathway are closely related to the occurrence and development of cardiovascular and neurological diseases.Although PKG has been widely studied,its complex functions in different physiological systems and potential innovative applications still need to be further explored.This article reviews the purification techniques for PKG,discusses the advantages and disadvantages of different extraction methods,summarizes the structure and activation mechanism of each domain of PKG,and analyzes the physiological functions of PKG in organisms,especially the well-established roles in the cardiovascular system,nervous system,and endocrine system.The emerging therapeutic applications of PKG are also reviewed.In addition,the challenges of this field are proposed at the end.
基金Supported by Youth Foundation of Education Department in Sichuan Province (07ZB060)Youth Science and Technology Innovation Fund in Sichuan Agricultural University~~
文摘[Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 pathogenic Escherichia coli in musk deer were extracted by the Lysis Triton method, and then identified by single enzyme digestion with three endonucleases of Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ. [Result] The yield rate of plasmids was 91.6%, and 24 pathogenic Escherichia coli in musk deer had the identical or similar plasmid profiles. [Conclusion] Plasmid DNA analysis offers scientific basis for molecular epidemiology of pathogenic Escherichia coli in musk deer in Sichuan Institute of Musk Deer Breeding.
