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
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
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
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
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
The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its...The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits.In this study,we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2.The non-essential elements were removed to create more stable derivatives pMUT1NR△and pMUT2HBC△.Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinaseⅢand the biosynthesis of ectoine.Compared to traditional antibiotic-dependent expression systems,our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.展开更多
Objective To investigate the effect of low concentration of Wenyang Tonglin Decoction(WTD)on the binding conditions of R45 plasmid conjugative transfer under liquid phase conjugation and its mechanism.Methods Escheric...Objective To investigate the effect of low concentration of Wenyang Tonglin Decoction(WTD)on the binding conditions of R45 plasmid conjugative transfer under liquid phase conjugation and its mechanism.Methods Escherichia coli CP9(R45)and Staphylococcus aureus RN450RF were cultured in medium containing WTD,and their minimum inhibitory concentration(MIC)values were obtained.Using promoter fusion technology,E.coli CP9(R45)containing a promoter fusion was obtained.β-Galactosidase activity of TrfAp and TrbBp was tested,and the mRNA expression of regulatory factors(TrbA,KorA,and KorB)was detected by real-time fluorescent quantitative polymerase chain reaction.Results The MIC of E.coli CP9(R45)was 400 g/L and that of S.aureus RN450RF was 200 g/L.When the drug concentration in the culture medium was 200 g/L,the highest number of conjugants was(3.47±0.20)×10^(7) CFU/mL At 90 h of conjugation,the maximum number of conjugants was(1.15±0.06)×10^(8) CFU/mL When the initial bacterial concentration was 10^(8) CFU/mL,the maximum number of conjugants was(3.47±0.20)×10^(7) CFU/mL.When the drug concentration was 200 g/L,theβ-galactosidase activity of TrfAp and TrbBp significantly increased;the relative quantification of TrbA,KorA and KorB were significantly inhibited.Conclusion Low concentration of WTD promoted the development of bacterial resistance by affecting promoters and inhibiting the expression of regulatory factors.展开更多
Polymyxins are a class of last-resort antibiotics used to treat infections caused by multidrug-resistant bacteria. Mobile resistance determinants for polymyxins, including mcr-1 to mcr-10 genes, have previously been r...Polymyxins are a class of last-resort antibiotics used to treat infections caused by multidrug-resistant bacteria. Mobile resistance determinants for polymyxins, including mcr-1 to mcr-10 genes, have previously been reported. Among them, mcr-10 has commonly beenobserved in Enterobacter and Klebsiella species. However, the presence of mcr-10 in Escherichia coli, an important opportunistic pathogen, has rarely been reported. This work describes the observation of mcr-10.1 in two clinical E. coli strains, with mcr-10.1 hosted ontwo novel plasmids, one of which carries transconjugation genes. These strains were isolated from anal fistula–suffering patients, suggesting their close relation to human bacterial infection. The genetic context of mcr-10.1 was also found to differ from those previouslyidentified in E. coli strains. This work is the first observation of mcr-10.1–carrying E. coli in clinical settings, expanding our knowledge ofthis important antibiotic resistance gene.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
A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB inser...A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.展开更多
Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is u...Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.展开更多
Since the first discovery of cyanobacterian plasmids in 1973, more than 60 strains have been found to have extra-chromosomal DNA, and their distribution covers almost all groups of the cyanobacteria. However, they wer...Since the first discovery of cyanobacterian plasmids in 1973, more than 60 strains have been found to have extra-chromosomal DNA, and their distribution covers almost all groups of the cyanobacteria. However, they were not discovered in the two unicellular genera Gloeocapsa and Gloeothece, and no further report was there on them ever since. Some species of these two genera having the ability to fix atmospheric nitrogen aerobically carry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
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
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.
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.
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financially supported by the National Natural Science Foundation of China(32370066,32000058)the Fundamental Research Funds for the Central Universities(JUSRP622003)+1 种基金National First-class Discipline Program of Light Industry Technology and Engineering(QGJC20230202)the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX23_2487).
文摘The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits.In this study,we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2.The non-essential elements were removed to create more stable derivatives pMUT1NR△and pMUT2HBC△.Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinaseⅢand the biosynthesis of ectoine.Compared to traditional antibiotic-dependent expression systems,our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.
基金Supported by the National Natural Science Foundation of China(No.81873070)。
文摘Objective To investigate the effect of low concentration of Wenyang Tonglin Decoction(WTD)on the binding conditions of R45 plasmid conjugative transfer under liquid phase conjugation and its mechanism.Methods Escherichia coli CP9(R45)and Staphylococcus aureus RN450RF were cultured in medium containing WTD,and their minimum inhibitory concentration(MIC)values were obtained.Using promoter fusion technology,E.coli CP9(R45)containing a promoter fusion was obtained.β-Galactosidase activity of TrfAp and TrbBp was tested,and the mRNA expression of regulatory factors(TrbA,KorA,and KorB)was detected by real-time fluorescent quantitative polymerase chain reaction.Results The MIC of E.coli CP9(R45)was 400 g/L and that of S.aureus RN450RF was 200 g/L.When the drug concentration in the culture medium was 200 g/L,the highest number of conjugants was(3.47±0.20)×10^(7) CFU/mL At 90 h of conjugation,the maximum number of conjugants was(1.15±0.06)×10^(8) CFU/mL When the initial bacterial concentration was 10^(8) CFU/mL,the maximum number of conjugants was(3.47±0.20)×10^(7) CFU/mL.When the drug concentration was 200 g/L,theβ-galactosidase activity of TrfAp and TrbBp significantly increased;the relative quantification of TrbA,KorA and KorB were significantly inhibited.Conclusion Low concentration of WTD promoted the development of bacterial resistance by affecting promoters and inhibiting the expression of regulatory factors.
文摘Polymyxins are a class of last-resort antibiotics used to treat infections caused by multidrug-resistant bacteria. Mobile resistance determinants for polymyxins, including mcr-1 to mcr-10 genes, have previously been reported. Among them, mcr-10 has commonly beenobserved in Enterobacter and Klebsiella species. However, the presence of mcr-10 in Escherichia coli, an important opportunistic pathogen, has rarely been reported. This work describes the observation of mcr-10.1 in two clinical E. coli strains, with mcr-10.1 hosted ontwo novel plasmids, one of which carries transconjugation genes. These strains were isolated from anal fistula–suffering patients, suggesting their close relation to human bacterial infection. The genetic context of mcr-10.1 was also found to differ from those previouslyidentified in E. coli strains. This work is the first observation of mcr-10.1–carrying E. coli in clinical settings, expanding our knowledge ofthis important antibiotic resistance gene.
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the National Natural Science Foundation of China and Microbial Resource Project of the Ministry of Science and Technology of China (Grant No. 2005DKA21208-6)
文摘A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.
文摘Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.
文摘Since the first discovery of cyanobacterian plasmids in 1973, more than 60 strains have been found to have extra-chromosomal DNA, and their distribution covers almost all groups of the cyanobacteria. However, they were not discovered in the two unicellular genera Gloeocapsa and Gloeothece, and no further report was there on them ever since. Some species of these two genera having the ability to fix atmospheric nitrogen aerobically carry
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 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.
