Differential amplification efficiency of pMB1 and p15A (ColE1-type) replicons in Escherichia coli stringent and relaxed strains starved for particular amino acids

Abstract

It was demonstrated previously that ColE1-type plasmids, the most commonly used vectors in molecular cloning, can be amplified in amino acid-starved relA mutants of Escherichia coli. Subsequent studies demonstrated that replication of at least some plasmids during amino acid starvation depends not only on the host relA allele but also on temperature and on the nature of deprived amino acid. Therefore, we investigated efficiency of amplification of two types of ColE1 plasmids (pMB1- and p15A-derived replicons) in E. coli relA+ and relA- hosts starved for different amino acids at 30 degrees C, 37 degrees C and 43 degrees C. We found differential amplification efficiency of plasmids pBR328 (pMB1-derived replicon) and pACYC184 (p15A-derived replicon) in the relA mutant during starvation for particular amino acids. Although amplification of pBR328 was negligible in the relA+ host, significant increase in pACYC184 content was observed in this strain starved for some (but not all) amino acids. The amplification efficiency of pBR328 and pACYC184 was found to be dependent on temperature. These results indicate that for maximal amplification of particular plasmid appropriate amino acid starvation and optimal temperature should be chosen. Our findings are in agreement with recently proposed model of the regulation of ColE1-type plasmid replication in amino acid-starved E. coli cells.