Infectious Nucleic Acids of Escherichia coli Bacteriophages

Various strains of Escherichia coli were grown to a titer of 5 × 10 8 cells/ml in peptone media. Following osmotic shocks (peptone → water → 0.4 M sucrose) or lysozyme‐EDTA treatment in hypertonic media, the spheroplasts were stored at 4° and then tested for competence with ØX 174 and M 13 phage DNA and M 12 phage RNA. Lysozyme spheroplasts proved most effective and yielded the following maximum infectivities: for one Ø× 174 DNA molecule, 5 × 10 −3 infectious DNA centers; for one M 13 DNA molecule, 10 −5 infectious DNA centers; and for one M 12 phage RNA molecle, 2 × 10 −5 infectious RNA centers. For many spheroplast preparations competence remained nearly constant for one week. One strain, E. coli 112–12 (λh)F + , retained a constant (though low) level of competence for months when frozen in 10% (v/v) dimethyl sulfoxide. Ø× 174 DNA saturation experiments indicated that 0.05–1% of the lysozyme spheroplasts were competent. Between 12 and 20 minutes after the addition of a non‐saturating level of 32 P‐labelled Ø× 174 DNA to such competent cell populations at 37°, 10–20% of the label was adsorbed. Noncompetent cell populations adsorbed no more than 4% of the added label. Prolonging the DNA adsorption time did not give a higher efficiency of DNA infection, because the cells began losing competence through a temperature‐dependent process. More than 90% of the infectious Ø× 174 DNA centers reamined sensitive to DNAase during the first 20 minutes in DNA adsorption medium. Plating Ø× 174 DNA and M 12 RNA‐infected cells on eosin methylene blue agar during this period prevented the formation of phage, whereas the plaque‐forming ability of the mature phage was not inhibited. These observations may suggest an unusual permeability of the competent cells. Inhibitors of phage RNA infection were removed by washing the competent cells. DNAase pretreatment of the spheroplasts increased their competence for infectious M 12 phage RNA.