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A New Method For Direct Selection Of Plasmid‐free Segregants Using Mercury Hypersensitivity As A Phenotypic Marker Of Bacterial Plasmids

T. Mori, T. Kaga, K. Toda, H. Nakahara, H. Ohtake
Published 1991 · Chemistry, Medicine

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A new method was developed for direct selection of plasmid‐free segregants using mercury hypersensitivity (Hgss) as a phenotypic marker of bacterial plasmids. The Hgss 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 Hgss phenotype. The Hgss marker was introduced into high‐copy‐number plasmids pUC18, pBR322, and pHSG298. Segregational loss of the Hgss plasmids caused a significant increase of resistance to Hg2+, and this allowed direct selection of plasmid‐free segregants on nutrient agars containing 1–2 μg HgCl2 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 Hgss marker proved to be useful for direct selection of plasmid‐free segregants from a mixed population of plasmid‐harboring and plasmid‐free cells.
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