The bacteriophage studied was T4 (ATCC 11303-B4), and its host bacteria were Escherichia coli (ATCC 11303).
The preparation of T4 virions:
Peptone broth (peptone 10 g, glucose 1 g, NaCl 3 g, 0.1M CaCl2 1 ml, 0.1M MgCl2 10 ml, 0.1M KH2PO4 3.2 ml, in 1 l solution, pH 7.2) was used for culturing the host bacteria, E. coli. T4 suspension was obtained by the plate lysate method and the small-scale liquid culture . In the plate lysate method, the virions were extracted by adding 2–3 ml of an electrolyte solution (EL) including 1.8 mM NaCl, 0.12 mM MgSO4, 0.12 mM MgCl2, 0.034 mM CaCl2 and 0.05 mM KCl, incubated several hours to elute virions and finally filtrate the eluent with 0.2 µm filter (Advantec AS020) to remove bacteria and bacterial debris. To exclude the effects of anonymous ions and ATP in the packaging experiments, the suspensions of T4 virions were purified with ultracentrifugation and dialysis. For ultracentrifugation, crude bacteriophage particles were purified by isopycnic centrifugation through CsCl gradients [15; Beckman XPN-90, SW32 rotor, 4 ℃, 24 h]. Following to ultracentrifugation, T4 suspensions were dialysed with Nuclepore polycarbonate membrane filters (0.015 µm in pore size, Whatman Inc.) against 0.5 mM CaCl2 for one week replacing the outside 0.5 mM CaCl2 five times. In the case to remove ions in viral particles, the viral suspensions were dialyzed against Milli-Q water for one week replacing outside water twice a day. The chemicals used were special grade products from Wako Pure Chemical Ind. Ltd.
Plaque forming unit (pfu):
Plaque forming units (pfu) were measured by plating aliquot of virions, adjusted to 10 ~ 500 plaques per plate as possible, on 1% agar peptone plates and 0.5% agar peptone top agar. Inoculated plates were incubated at 36℃ for 12 hours before counting. The pfu values of T4 samples were enumerated frequently to confirm the activity of the T4 virion during the experiments.
Fluorescent light microscopic observation (FLM):
Virion particles and ejected DNA were distinguished with fluorescent microscopy. Aliquots of the suspensions of virions and DNA were collected on 0.02 µm Anodisc (ø25 mm), backed by a pre-moisturized filter (Millipore HA). Virions and DNA collected on the filters were stained with the filtration of ca. 30 µl of 0.001 dilution of SYBR-Gold (Molecular Probes, Inc.) including 0.1 or 10 mM phosphate buffer (pH 8.0), 0.1 mM EDTA (pH 8.0) and 50 mM dithiothreitol (Wako Pure Chemical Ind. Ltd. for molecular biology). Sample filters were mounted on a glass slide with a mounting medium composed with 50% glycerol (Wako Pure Chemical Ind. Ltd., Special grade), 0.1 or 10 mM phosphate buffer (pH 8.0) and 40 mM dithiothreitol. The phosphate buffer concentration was selected in accordance with the viral specimen, i.e. 10 mM for ejected DNA and 0.1 mM for compact DNA. In addition, 10 mM MgCl2 were added for the specimen of compact DNA. Samples were observed with Olympus BX50 epifluorescent microscope, equipped with N.A. 1.35 UPlan Apo x100 objective lens, N.A. 0.4 UPlan Apo x10 objective lens and U-MWBV dichroic mirror unit. At the beginning of enumeration, the even distribution of the specimens on a filter was confirmed by a general view of the whole filter. Enumerations were carried out at randomly selected more than 20 fields along a diameter from one periphery to another periphery. Total numbers of the counted objects were more than 200, except for the cases where were almost no object.
Ejection of T4 DNA:
The ejection of DNA from T4 virion was examined with biological (Pi, citrate and an electrolyte mimicking cell sap) and an artificial (EDTA, Tris-HCl and TE (Nippon Gene Co., LTD)) chelates (pH 8.0). The composition of an electrolyte mimicking cell sap is; 15 mM Na+, 140 mM K+, 0.1 mM Mg2+, 10 mM Cl-, 10 mM HCO3-, 35 mM HPO42- [17,18]. The virions were exposed to combinations of counterion concentrations, i.e. phosphate buffer (pH 7.6) including 0.01–100 mM Pi vs. 0–1 mM CaCl2 or 0–1 mM Mg Cl2, for several minutes.
Packaging of T4 DNA:
The packaging of DNA into the capsids of T4 was induced by 10–105 times dilution of the ejected specimens. The solvent used for the dilution were 1 mM CaCl2 solution or EL. The diluted suspensions of T4 virions were incubated for ca. 10 min. The total number of reformed virions were measured visually by FLM and their infective ability was estimated by pfu counts of the plate method.
DNase I treatment:
DNase I (recombinant DNase I, Takara) treatment was applied to discriminate the DNA covered with capsid proteins from the naked DNA without capsid coverings. Two types of DNase I digestions were proceeded. One was DNase I degradations in situ. DNase I was added to suspensions of T4 in ES, 30 mM pi and TE with the concentrations of 1 U/40 µl plus 1 mM MnCl2. The specimens were incubated for 2 hours.
The other treatments were the on-filter degradation. Suspensions of virions are filtrated on 0.02 µm Anodisc (ø25 mm), followed by mounting of DNase I solution, ca. 0.1–0.2 ml of 1 U/40 µl dilution plus 1 mM MnCl2  in EL, on the filter to degrade naked DNA. This process removes the original solvent to replace DNase I solution. The DNase treatments were incubated for 0.5–1 hour.
After incubation, specimens were filtrated and stained with SYBR-Gold.
Digestion of capsid proteins by proteinase K:
To obtain naked DNA molecules of T4 free from capsids, the capsid proteins of viral suspension was digested with proteinase K (Takara, ≥ 600 mAnson U/ml) . Virions were suspended in EL, pH 8.0, with 1% v/v of proteinase K, which was incubated at room temperature for one day. Tris, EDTA and SDS, which were normally included in the lysis buffer, were not included in the present mixture of proteinase K degradation in order to avoid any negative effects on pfu (This will be discussed in the text). Inactivated proteinase K, heated the enzyme solution at 95℃ for 15 min, was used as the control of the proteinase K treatment.
The concentration of ATP was measured with triplicate specimens. The total concentration of ATP + ADP + AMP between 10 pM and 10 µM was measured by a reagent set, Lucipack A3 water, and Lumitester Smart (Kikkoman Biochemifa Co.). Hereinafter, the total amount of ATP, ADP and AMP is indicated as ATP concentration