Adenosine monophosphate-induced amplification of ColE1 plasmid DNA in Escherichia coli.
ColE1 plasmid copy number was analyzed in relaxed (relA) and stringent (relA(+)) Escherichia coli cells after supplementation of culture media with adenosine monophosphate (AMP). When a relaxed E. coli strain bearing ColE1 plasmid was cultured in LB medium for 18 h and induced with AMP for 4h, the plasmid DNA yield was significantly increased, from 2.6 to 16.4 mgl(-1). However no AMP-induced amplification of ColE1 plasmid DNA was observed in the stringent host. Some plasmid amplification was observed in relA mutant cultures in the presence of adenosine, while adenine, ADP, ATP, ribose, potassium pyrophosphate and sodium phosphate caused a minor, if any, increase in ColE1 copy number. A mechanism for amplification of ColE1 plasmid DNA with AMP in relA mutant bacteria is suggested, in which AMP interferes with the aminoacylation of tRNAs, increases the abundance of uncharged tRNAs, and uncharged tRNAs promote plasmid DNA replication. According to this proposal, in relA(+) cells, the AMP induction could not increase ColE1 plasmid copy number because of lower abundance of uncharged tRNAs. Our results suggest that the induction with AMP can be used as an effective method of amplification of ColE1 plasmid DNA in relaxed strains of E. coli.
Wang
Zhijun
Z
Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, PR China.
Xiang
Li
L
Shao
Junjie
J
Wegrzyn
Grzegorz
G
D000249:Adenosine Monophosphate / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D004261:DNA Replication; D004269:DNA, Bacterial / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D005784:Gene Amplification / Q000187:drug effects*; D010957:Plasmids / Q000235:genetics*; D012329:RNA, Bacterial / Q000187:drug effects / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; C042833:ROM protein, Bacteria; D000249:Adenosine Monophosphate