An auxotrophic strain of E. coli K12 treated with CaCl2 was transformed for several markers at a frequency of up to 10-6 per recipient cell by a DNA preparation isolated from a prototrophic strain. The transforming activity of the DNA preparation was eliminated by treatment with DNase, heat, or sonication, whereas RNse or Pronase treatment had little effect. Two closely linked genetic markers (leu and ara) showed a high degree of cotransformation linkage when high molecular weight DNA was used, but the linkage was almost completely eliminated when sheared, smaller molecular weight DNA was used. There is genetic evidence that the transformation is a result of the replacement of the preexisting genetic marker on the chromosome by that of the donor DNA.Since its discovery in Pneumococcus (1, 2), genetic transformation has been reported among several other bacterial species such as Haemophilus influenzae (3) and Bacillus subtilis (4). In Escherichia coli, especially in its K12 strain, however, attempts to demonstrate genetic transformation have been unsuccessful. Several unique systems are known in E. coli in which the biological activity of isolated DNA can be assayed directly, such as transformations by bacteriophage X DNA with helper phages (5). More recently, an efficient transfection system was developed by Mandel and Higa using CaClrtreated cells (6). In most examples of transfection and transformation, the DNA is either phage or plasmid (7) DNA, which is circular (or converts to circular forms in the cells).During studies on the mechanism of ATP-dependent DNase (8-11) from E. coli in this laboratory, we have observed that the enzyme does not attack double-stranded circular DNA with or without nicks, whereas it causes extensive degradation of linear DNA molecules. Since fragments of bacterial chromosome are the source of genetic material in other general transformation systems, one of the reasons for the failure of genetic transformation in E. coli cells might be extensive degradation by the enzyme of linear DNA molecules lacking any protection mechanism. These characteristics of the enzyme plus reports of a mutant that lacks ATP-dependent DNase yet retains the capacity for genetic recombination (12) prompted us to examine the possibility of genetic transformation in E, coli K12. In this paper, we report the genetic transformation of E. coli K12 and some of its basic characteristics.
MATERIALS AND METHODSBacterial Strains used in this study are listed in Table 1. Genetic symbols are those used by Taylor (13).Reagents. RNase (pancreas, crystalline RNase A), DNase (pancreas, crystalline), and calf-thymus DNA were purchased from Worthington Biochemical Co. Pronase (grade B) was purchased from Calbiochem.Isolation of Transforming DNA. Cells were grown in Pen assay broth (Difco), chilled, and harvested at the late exponential or early stationary phase by centrifugation. They were then suspended in one-fortieth of the original volume of cold 0.1 M NaCl-0.1 M EDTA (pH 8.5). 1 M Tris HCl (pH 9.0)-10% SDS (sodi...