Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA

Abstract

Transformation of E. coli cells treated with CaCJ2 to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before antibiotic challenge. MATERIALS AND METHODSBacterial Strains and R Factors. The I-like R factor, R64-11 (16), which specifies resistance to tetracycline (Tc) and streptomycin (Sm), was obtained from R. Curtiss. R6 (17), an F-like R factor that carries resistance to kanamycin (Km), neomycin (Nm), chloramphenicol (Cm), sulphonamide (Su), streptomycin, and tetracycline, was obtained from T. Watanabe. R6-5, a spontaneous variant of R6 that lacks tetracycline resistance, was isolated in our laboratory (18). The bacterial strains used in these experiments have been described (7,22). DNA Preparations. In certain instances, covalently-closed R-factor DNA was isolated and purified from E. coli as described (6, 7). Alternatively, a Brij-lysis procedure (19) was used for initial R-factor DNA isolation, and preparations obtained by this method were subsequently purified by centrifugation in cesium chloride-ethidium bromide gradients. The catenated, closed circular, and noncircular forms of R-factor DNA used in experiments comparing the relative transforming ability of the various R-factor DNA species were isolated from E. coli minicells. R-factor DNA was denatured by heating it at 980 for 5 min in 15 mM NaCl-1.5 mM Na citrate followed by rapid cooling at 0°. Sonication of R-factor DNA to about 9S fragments was done for 15 see at 00 by a Branson model W185 D sonicator, and the size of the R-factor DNA fragments was confirmed by sucrose gradient centrifugation (7).Transformation Reaction Mixture. Transformation was done by a variation of the procedure of Mandel and Higa (15), as modified by Lobban, Masuda, and Kaiser (personal communication). E. coli strain C600 was grown at 370 in H1 medium (20) to an optical density of 0.85 at 590 nm. At this point, the cells 2110 Abbreviation: R factor, antibiotic resistance factor. * The previous paper in this series is ref. 18.

Destabilized inheritance of pSC101 and other Escherichia coli plasmids by DpiA, a novel two‐component system regulator

“…Transformation assays, segregation rates and plasmid copy number Cells containing the par þ pSC101 derivative pDLC8 (Conley and Cohen, 1995) were transformed using standard procedures (Cohen et al, 1972) with plasmids expressing dpiA and plated on LB plates containing antibiotics selective for either the incoming or both plasmids. Plasmid stability and copy number were determined as described by Meacock and Cohen (1980) and Biek and Cohen (1986), respectively, or by gene dosage of kanamycin resistance (Nordströ m et al, 1980).…”

Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids

“…DNA fragments were purified from gels by the crush-and-soak method (34). Transformation of E. coli was done by the method of Cohen et al (9).…”

Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

“…The resulting cDNA preparation was then ligated by EcoRI adaptors and cloned with T4 DNA ligase (Promega) into a plasmid pUC18 (Sangon, China), which had been cut with EcoRI (BRL) and treated with alkaline phosphatase (Takara, Japan) [31]. Transformation to E. coli DH5a was carried out by the calcium shock method described by Lederberg and Cohen [32,38].…”