Molecular Recombination Between R-Factor Deoxyribonucleic Acid Molecules in Escherichia coli Host Cells

Abstract

Three previously studied R factors were used: 222/R4, controlling transmissible resistance to sulfonamide, streptomycin, chloromycetin, and tetracycline (SU r SM r CM r TC r ); 222/R3, a derivative of 222/R4 (now termed 222/R3W) having lost TC r ; and R15, controlling infectious resistance to SU and SM only. Two types of derivative R factors were isolated from 222/R4 by serial subculture in Salmonella species. One derivative, termed 222/R1, lost resistance to SU, SM, and CM, and the other, termed 222/R3N, lost only TC r . Each factor was transferred to a standard Escherichia coli K-12 host. Recombinant factors of 222/R4 phenotype were isolated by selection after mixed culture of E. coli (222/R1) + and (222/R3N) + strains. Density-gradient equilibrium centrifugation of lysates of E. coli R + hosts in the presence of ethidium bromide separated R-factor deoxyribonucleic acid (DNA) as a heavy satellite peak which was subjected to electron microscopy or analytical density gradient centrifugation. Each DNA comprised a unimolecular species of circular DNA. The contour of R15 measured 22.3 μm [equivalent to 46 × 10 6 atomic mass units (AMU)], and that of 222/R4 measured 33.6 μm (70 × 10 6 AMU). 222/R3W appeared to be a point mutant or small deletion of 222/R4 with an almost identical size, whereas 222/R3N had lost a DNA segment of about 3 μm, and measured 30.3 μm or 63 × 10 6 AMU. The 222/R1 factors also appeared to have arisen by loss of DNA from 222/R4, 222/R1A being 22.3 μm or 46 × 10 6 AMU, whereas all other 222/R1 factors appeared to be duplicates, measuring 25.6 μm or 53 × 10 6 AMU. The DNA from six recombinant factors of R4 phenotype was indistinguishable in size and configuration from the parental 222/R4. In most cases, the number of R-factor copies (present as covalently closed circular molecules) per copy of the E. coli chromosome was less than 2, ranging from 1.2 to 3.3.