Molecular Structure of an R Factor, Its Component Drug-Resistance Determinants and Transfer Factor

Abstract

Plasmid DNA from Escherichia coli strains harboring drug resistance either of the infectious or noninfectious kind has been separated by CsCl centrifugation of crude cell lysates in the presence of ethidium bromide and examined by electron microscopy. Plasmid deoxyribonucleic acid (DNA) from an S + strain (which has the property of noninfectious streptomycin-sulfonamide resistance) consists of a monomolecular covalently closed circular species of 2.7 μm in contour length (5.6 × 10 6 atomic mass units; amu). DNA from a strain carrying a transfer factor, termed Δ, but no determinant for drug resistance, is a monomolecular covalently closed circular species of 29.3 μm in contour length (61 × 10 6 amu). DNA from either Δ + A + or Δ + S + strains, (which are respectively ampicillin or streptomycin-sulfonamide resistant, and can transfer this drug resistance), shows a bimodal distribution of molecules of contour lengths 2.7 μm and 29.3 μm, whereas DNA from a (Δ-T) + strain (showing infectious tetracycline resistance) contains only one species of molecule measuring 32.3 μm (67 × 10 6 amu). We conclude that ampicillin resistance is carried by a DNA molecule (the A determinant) of 2.7 μm, and streptomycin-sulfonamide resistance is carried by an independent molecule (the S determinant) of similar size. These molecules are not able to effect their own transfer, but can be transmitted to other cells due to the simultaneous presence of the transfer factor, Δ, which also constitutes an independent molecule, of size 29.3 μm. In general, there appears to be little recombination or integration of the A or S molecules into that of Δ, although a small proportion (5–10%) of recombinant molecules cannot be excluded. In contrast, the third drug-resistance determinant, that for tetracycline resistance (denoted as T), is integrated in the Δ molecule to form the composite structure Δ-T of size 32.3 μm, which determines infectious tetracycline resistance. The Δ + A + and Δ + S + strains are defined as harboring plasmid aggregates , and the (Δ-T) + strain is defined as carrying a plasmid cointegrate ; the properties of all three strains are characteristic of strains harboring R factors. These results are compatible with the previously published genetic data. The number of Δ molecules per cell appears to be equal to the chromosomal number irrespective of growth phase, and this plasmid can thus be defined as stringently regulated in DNA replication. In contrast, S and A exist as multiple copies, probably in at least a 10-fold excess of chromosomal copy number. S and A can thus be defined as relaxed in the regulation of their DNA replication.