Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarct...Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarctic bacteria, and developed a transposon Tn5 mutagenesis system. A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station, King George Island. Six Psychrobacter strains were identified as psychrophilic, with optimal growth tempera- tures of 10~C or 15~C Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4~C was selected for transposon mutagenesis. Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker. Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants, and for a single recombinant, Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807. The result indicates that the mutagenesis method, although successful, has a relatively low rate. The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms, and the mutagenesis method can be applied to genetic analysis.展开更多
Expression-independent gene or polyadenylation[poly（A）]trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed.Although a number of poly（A）-trap vectors have been...Expression-independent gene or polyadenylation[poly（A）]trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed.Although a number of poly（A）-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome,further efforts are needed to avoid the 3＇-terminal insertion bias and the splice donor（SD） read-through,and to improve the mutagenicity.Here,we present a Sleeping Beauty（SB） transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding,gene-breaking and large-scale mutagenesis, respectively.The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element（ARE）,which greatly reduced the SD read-through events,except that the internal ribosomal entry site（IRES） element was introduced in front of the SD signal to overcome the phenomenon of 3＇-bias gene trapping.The breaking cassette consisting of an enhanced splicing acceptor（SA）,a poly（A） signal coupled with a transcriptional terminator（TT） effectively disrupted the transcription of trapped genes.Moreover,the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11,which allows the conditional remobilization of a trap insert from a non-coding region.The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells.In addition,the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus,this poly（A）-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.展开更多
Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria’s high-level of intrinsic and acquired resistance to antibiotics. To address this p...Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria’s high-level of intrinsic and acquired resistance to antibiotics. To address this problem, it is crucial to investigate the molecular mechanisms of antibiotic resistance in this organism. In this study, a P. aeruginosa transposon insertion library of 17000 clones was constructed and screened for altered susceptibility to seven antibiotics. Colonies grown on agar plates con- taining antibiotics at minimum inhibitory concentrations (MICs) and those unable to grow at ? MIC were collected. The transposon-disrupted genes in 43 confirmed mutants that showed at least a three-fold increase or a two-fold decrease in suscep- tibility to at least one antibiotic were determined by semi-random PCR and subsequent sequencing analysis. In addition to nine genes known to be associated with antibiotic resistance, including mexI, mexB and mexR, 24 new antibiotic resis- tance-associated genes were identified, including a fimbrial biogenesis gene pilY1 whose disruption resulted in a 128-fold in- crease in the MIC of carbenicillin. Twelve of the 43 genes identified were of unknown function. These genes could serve as targets to control or reverse antibiotic resistance in this important human pathogen.展开更多
基金supported by the Korea Polar Research Institute(Grant nos.PE08050 and PE13240)
文摘Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarctic bacteria, and developed a transposon Tn5 mutagenesis system. A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station, King George Island. Six Psychrobacter strains were identified as psychrophilic, with optimal growth tempera- tures of 10~C or 15~C Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4~C was selected for transposon mutagenesis. Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker. Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants, and for a single recombinant, Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807. The result indicates that the mutagenesis method, although successful, has a relatively low rate. The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms, and the mutagenesis method can be applied to genetic analysis.
基金supported by the National Natural Science Foundation of China(No.30871442) to Z.Cuithe National Basic Research Program of China(No.2012CB944500)
文摘Expression-independent gene or polyadenylation[poly（A）]trapping is a powerful tool for genome-wide mutagenesis regardless of whether a targeted gene is expressed.Although a number of poly（A）-trap vectors have been developed for the capture and mutation of genes across a vertebrate genome,further efforts are needed to avoid the 3＇-terminal insertion bias and the splice donor（SD） read-through,and to improve the mutagenicity.Here,we present a Sleeping Beauty（SB） transposon-based vector that can overcome these limitations through the inclusion of three functional cassettes required for gene-finding,gene-breaking and large-scale mutagenesis, respectively.The functional cassette contained a reporter/selective marker gene driven by a constitutive promoter in front of a strong SD signal and an AU-rich RNA-destabilizing element（ARE）,which greatly reduced the SD read-through events,except that the internal ribosomal entry site（IRES） element was introduced in front of the SD signal to overcome the phenomenon of 3＇-bias gene trapping.The breaking cassette consisting of an enhanced splicing acceptor（SA）,a poly（A） signal coupled with a transcriptional terminator（TT） effectively disrupted the transcription of trapped genes.Moreover,the Hsp70 promoter from tilapia genome was employed to drive the inducible expression of SB11,which allows the conditional remobilization of a trap insert from a non-coding region.The combination of three cassettes led to effective capture and disruption of endogenous genes in HeLa cells.In addition,the Cre/LoxP system was introduced to delete the Hsp70-SB11 cassette for stabilization of trapped gene interruption and biosafety. Thus,this poly（A）-trap vector is an alternative and effective tool for identification and mutation of endogenous genes in cells and animals.
基金supported by the National Natural Science Foundation of China (Grant Nos. 30870097 and 30611120520)
文摘Pseudomonas aeruginosa causes acute and chronic infections in humans and these infections are difficult to treat due to the bacteria’s high-level of intrinsic and acquired resistance to antibiotics. To address this problem, it is crucial to investigate the molecular mechanisms of antibiotic resistance in this organism. In this study, a P. aeruginosa transposon insertion library of 17000 clones was constructed and screened for altered susceptibility to seven antibiotics. Colonies grown on agar plates con- taining antibiotics at minimum inhibitory concentrations (MICs) and those unable to grow at ? MIC were collected. The transposon-disrupted genes in 43 confirmed mutants that showed at least a three-fold increase or a two-fold decrease in suscep- tibility to at least one antibiotic were determined by semi-random PCR and subsequent sequencing analysis. In addition to nine genes known to be associated with antibiotic resistance, including mexI, mexB and mexR, 24 new antibiotic resis- tance-associated genes were identified, including a fimbrial biogenesis gene pilY1 whose disruption resulted in a 128-fold in- crease in the MIC of carbenicillin. Twelve of the 43 genes identified were of unknown function. These genes could serve as targets to control or reverse antibiotic resistance in this important human pathogen.
