A new method for direct selection of plasmid‐free segregants using mercury hypersensitivity as a phenotypic marker of bacterial plasmids

Abstract

A new method was developed for direct selection of plasmid-free segregants using mercury hypersensitivity (Hg(ss)) as a phenotypic marker of bacterial plasmids. The Hg(ss) marker originated from the 4.8-kb EcoRI fragment H of the R-factor R100. Since the EcoRI fragment H spans the majority of the mercury resistance operon (mer), but lacks the intact merA gene coding for the mercury reductase enzyme, this fragment conferred the Hg(ss) phenotype. The Hg(ss) marker was introduced into high-copy-number plasmids pUC18, pBR322, and pHSG298. Segregational loss of the Hg(ss) plasmids caused a significant increase of resistance to Hg(2+), and this allowed direct selection of plasmid-free segregants on nutrient agars containing 1-2 mug HgCl(2) ml(-1). Plasmid-loss segregants were estimated to appear at frequencies ranging from 10(-3) to 10(-7) for the tested high-copy-number plasmids. THe Hg(ss) marker proved to be useful for direct selection of plasmid-free segregants from a mixed population of plasmid-harboring and plasmid-free cells.