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A new method for direct selection of plasmid‐free segregants using mercury hypersensitivity as a phenotypic marker of bacterial plasmids
Author(s) -
Mori Tsukasa,
Kaga Takayuki,
Toda Kiyoshi,
Nakahara Hideomi,
Ohtake Hisao
Publication year - 1991
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260370211
Subject(s) - plasmid , ecori , biology , pbr322 , operon , genetics , microbiology and biotechnology , gene , recombinant dna , phenotype , escherichia coli
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 μg 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.