Curing of Bacillus subtilis plasmid using sodium dodecyl sulfate (SDS)was studied in order to obtain a host strain. An overnight culture of Bacillus subtilis 24/pMX45 was used to inoculate fresh LB containing SDS (0-0...Curing of Bacillus subtilis plasmid using sodium dodecyl sulfate (SDS)was studied in order to obtain a host strain. An overnight culture of Bacillus subtilis 24/pMX45 was used to inoculate fresh LB containing SDS (0-0.008%). No growth of 24/pMX45 was observed when LB contained an SDS concentration of 0.006% or greater, and the sublethal concentration (w/v) of SDS was 0.005% with a killing rate of 99%. Samples were diluted and plated on LB agar, individual colonies were randomly picked to a selective agar medium by tooth to screen for loss of plasmid-encoded erythomycin resistance. CsCl-EtBr gradient centrifugation and plasmid DNA profile demonstrated that plasmid-cured derivative A7 has completely lost its plasmid. A7 had a shorter lag, and its cell concentration was consistently higher than that of the 24/pMX45. Elimination of the plasmid was first observed after 24/pMX45 had been treated with SDS for 8 h. The percent elimination then continued to increase until about 22 h, after which the fraction of cured cell in the population remained constant. Plasmid cured cell numbers were measured in a separate control culture of 24/pMX45 untreated by SDS. No spontaneous loss of pMX45 was observed after 24/pMX45 were incubated for 24 h and 48 h with shaking at 37 ℃.These results suggested that SDS can be used as curing agent to eliminate the plasmid of Bacillus subtilis.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 insertion,...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
文摘Curing of Bacillus subtilis plasmid using sodium dodecyl sulfate (SDS)was studied in order to obtain a host strain. An overnight culture of Bacillus subtilis 24/pMX45 was used to inoculate fresh LB containing SDS (0-0.008%). No growth of 24/pMX45 was observed when LB contained an SDS concentration of 0.006% or greater, and the sublethal concentration (w/v) of SDS was 0.005% with a killing rate of 99%. Samples were diluted and plated on LB agar, individual colonies were randomly picked to a selective agar medium by tooth to screen for loss of plasmid-encoded erythomycin resistance. CsCl-EtBr gradient centrifugation and plasmid DNA profile demonstrated that plasmid-cured derivative A7 has completely lost its plasmid. A7 had a shorter lag, and its cell concentration was consistently higher than that of the 24/pMX45. Elimination of the plasmid was first observed after 24/pMX45 had been treated with SDS for 8 h. The percent elimination then continued to increase until about 22 h, after which the fraction of cured cell in the population remained constant. Plasmid cured cell numbers were measured in a separate control culture of 24/pMX45 untreated by SDS. No spontaneous loss of pMX45 was observed after 24/pMX45 were incubated for 24 h and 48 h with shaking at 37 ℃.These results suggested that SDS can be used as curing agent to eliminate the plasmid of Bacillus subtilis.
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.
