Physiological studies on pBR 322 DNA amplification in an Escherichia coli relA strain.

Abstract

Amino acid limitation leads in E. coli relA cells which cannot synthesize guanosine tetraphosphate (ppGpp) under these conditions to an amplification of pBR 322 DNA. We previously proposed that ppGpp produced in E. coli relA+ cells subjected to amino acid limitation inhibits pBR 322 DNA replication (Hecker et al. 1983). In further experiments it was established that an E. coli relA strain shows plasmid amplification during amino acid limitation (arginine, threonine, leucine or histidine) only in the presence of sufficient concentrations of phosphate, ammonia and glucose. Plasmid amplification does not occur if ammonia or phosphate is depleted. We suggest that glucose, ammonia and phosphate are needed for nucleotide and deoxynucleotide synthesis as the essential prerequisite for plasmid amplification. The activity of beta-lactamase was determined as an indicator for the expression of plasmid-encoded genes. The enzyme activity remains on a low level during plasmid amplification because of arginine exhaustion. A remarkable increase in the activity of beta-lactamase was observed after resumption of growth of relA cells containing amplified plasmid DNA. The plasmid content decreased as the cells continued to grow. We found that plasmid amplification and expression of plasmid-localized genes are opposite reactions which do not occur at the same time.