Fate of conjugally transferred DNA in minicells of Escherichia coli K-12

DNA-deficient minicells produced by an Escherichia coli F^- strain can act as recipients for conjugally transferred DNA. When single-stranded [³H] thymidine-labeled Hfr DNA was transferred to minicells, 40 to 50% of the DNA could be degraded at 37°C in 3 hr into cold, trichloroacetic-acid-soluble material. Most of the acid soluble radioactivity was identified as thymine. The rates and amounts of DNA degradation were dependent on the temperature of incubation and on the pH and ionic makeup of the medium in which the minicells were suspended. Degradation was stimulated by the presence of Mg²⁺, Mn²⁺, and especially Ca²⁺ and was inhibited by Na⁺ and the absence of divalent cations and by the presence of dinitrophenol. The rates and amounts of degradation were greater in minicells harvested from stationary-phase cultures than in minicells from log-phase cultures. The results indicate that the DNA was degraded enzymatically and that both exonucleolytic and endonucleolytic activities were present in the minicells. From an analysis of undegraded DNA in minicells by alkaline sucrose gradient centrifugation, we infer that degradation is an all-or-none affair. Further, since the rates and amounts of DNA degradation observed in DNA-deficient F^- minicells were similar to those in plasmid-containing minicells used as recipients for Hfr DNA, it can be inferred that the nucleases responsible for the degradation are cytoplasmic and are not bound to undamaged plasmid or chromosomal DNA.