EFFECTS OF THE PAR LOCUS ON THE GROWTH-RATE AND STRUCTURAL STABILITY OF RECOMBINANT CELLS

Abstract

A decreased growth rate of recombinant cells is observed when a cloned gene protein encoded in a multicopy plasmid is induced from a strong promoter. This negative effect on the cell growth rate may be the consequence of alternate use or reallocation of energy, precursor metabolites, and protein synthesizing machinery. In order to analyze the causes for this adverse effect, we have studied how genetic elements present in multicopy plasmids affect the growth of recombinant Escherichia coli (K12DELTAH1DELTAtrpEA). Turning on of the P(L) promoter, whether or not protein-coding sequences were present downstream of the promoter, decreased the growth of the recombinant cells by 15-50 %. This result suggests that the essential factor(s) for cell growth may be sequestered by the genetic elements present in the plasmids and thus may cause the decreased growth. The negative effect of the de-repressed P(L) promoters on cell growth was partially reversed when the par sequence from pSC101 was inserted into the same plasmid. In each case when the plasmid carried only the P(L) promoter, the P(L) promoter followed by the N-terminal part of the protein-coding region, or the P(L) promoter followed by complete sequences encoding a protein, the par sequence exhibited a positive effect on the growth rate of the recombinant cells, which usually grow at a slower rate than the host cell. In addition, it was found that the recombinant cells bearing the plasmid with the par locus were as resistant as the host cell to osmotic shock when exposed to distilled water, whereas the recombinant cells bearing the plasmid without the par locus were sensitive to osmotic shock and resulted in lysis.