Isolation and curing of the Klebsiella pneumoniae large indigenous plasmidusing sodium dodecyl sulphate

The ability of Klebsiella pneumoniae (NCTC, CL687/80) to produce and, in tu rn, excrete glutamate has been equated with the presence of a large indigen ous plasmid with an apparent molecular mass in the region of 96 +/- 2 kbp ( n = 6). Unlike mitomycin C, novobiocine and ethidium bromide (curing agents ), the use of sodium dodecyl sulphate (SDS) proved very effective in curing the plasmid with a relatively high frequency (6.25 x 10(-4)). Furthermore, the absence of isocitrate dehydrogenase (ICDH) activity in the cured strai n strongly suggests that the structural gene encoding ICDH in this organism , in sharp contrast to all known ICDHs, is plasmid-encoded. Moreover, the S DS-based protocol reported for the isolation of the K. pneumoniae indigenou s plasmid has proven successful with other organisms including Pseudomonad and Corynebacteria, as well as in recombinant strains of Escherichia coli. (C) 2000 Editions scientifiques et medicales Elsevier SAS.