Infectious Nucleic Acids of Escherichia coli Bacteriophages

Abstract

Various strains of Escherichia coli were grown to a titer of 5 x lo8 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 20 minutes after the addition of a non-saturating level of SAPlabelled @X 174 DNA to such competent cell populations at 37", 10-20°/, of the label was adsorbed. Noncompetent cell populations adsorbed no more than 4O//, 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 goo/, of the infectious @X 174 DNA centers remained sensitive to DNAase during the first 20 minutes in DNA adsorption medium. Plating @X 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.There has been a rapid succession of papers establishing the infectivity of a variety of phage DNA's and RNA for several hosts. These systems include @X 174-Escherichia coli bacteria [1,2,3,4] The procedures required for high infectious DNA yields vary with the type of host cell. In Bacillus subtilis and Haemophilus influenzae cultures many competent bacteria appear "spontaneously" during some stage of growth in special media. I n contrast, E. coli strains have a very low level of "spontaneous"Non-Standard Abbreviation. Plaque-forming units, p.f.u [7,12,13]. Osmotic shock of E . coli may also induce high levels of competence for infectious @X 174 DNA [I].In the present communication various methods of preparing competent E. coli bacteria are compared.Several improvements on earlier methods are also described; in particular, a preparation of frozen spheroplasts of constant competence is presented and methods for preparing spheroplasts of high

Filamentous bacterial viruses.

“…Single-stranded Ff DNA is infectious to spheroplasts prepared from either Fi or Fcells, although about 10-fold less so than 45X174 DNA (18,19,85,127,153). Thus, formation of RF from VS can take place in spheroplasts.…”

“…Infectious DNA can be isolated from FV by hot phenol or by phenol plus sodium dodecyl sulfate (SDS); further purification can be obtained by precipitation with alcohol (19,87,127,135,162). DNA can also be isolated by alkali treatment followed by banding in a CsCl density gradient (53,157).…”

Restriction of infectious bacteriophage fd DNA's and an assay for in vitro host-controlled restriction and modification.

“…3 Following exhaustive dialysis against 0.01 M Tris buffer, pH 8, containing 1 mM EDTA, the DNA was further purified by CsCl density gradient centrifugation.…”

Filamentous bacterial viruses

“…This effect may result from the low level of suppression by UAA suppressors (70), or it may result from a virus-induced alteration in the mechanism of translation (58) (19,87,127,135,162). DNA can also be isolated by alkali treatment followed by banding in a CsCl density gradient (53,157 (127) and was dialyzed at a concentration of about 300 jxg/ml against 0.2 M NaCI, 0.02 M tris (hydroxymethyl)aminomethane (Tris), 0.002 M ethylenediaminetetraacetic acid (EDTA) (pH 7.5) and then against 0.2 M NaCI, 0.02 M Tris (pH 7.5; dialysis tubing was presoaked in EDTA).…”