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Microbes & Immunity An ATP-free packaging of T4 DNA

Treatment with chelating agents, such as EDTA, induces during the process. To remove ions in viral particles, the
the ejection of DNA from the head of the bacteriophage viral suspensions were dialyzed with Spectro/Por 6 dialysis
8,9
and renders the phage activity unrecoverable afterward. 8-10 membrane, MWCO 1000, against T-buffer containing
Among the ions which create ion potential on the cell 100 mM NaCl, 2 mM MgSO , and 0.5 mM phosphate buffer
4
membranes essential for the activity for life, calcium (pH 7.5), for 1 week, with the T-buffer replaced twice a day.
11
ion (Ca ) and inorganic orthophosphate ion (Pi) form The chemicals utilized in this study were special grade
2+
the steepest gradients. We hypothesized that Ca and products from Wako Pure Chemical Ind. Ltd.
12
2+
Pi can be critical ions for the phage-host cell relationship.
Here, a system of reversible DNA ejection and packaging 2.3. Plaque-forming unit measurement
induced by adjustment of the ambient phosphate (chelate) Plaque-forming units (pfu) were measured in duplicate by
and Ca (divalent cation) concentrations mimicking the plating aliquot of virions, adjusted to 10 – 500 plaques per
2+
inside and outside of a cell, respectively, is introduced. As plate, on 1% agar peptone plates and 0.5% agar peptone
a result of ultracentrifugation, dialysis, and dilution, the top agar. For measuring the time course of pfu, a single
concentrations of ATP, when the packaging of the DNA plate was prepared repeatedly during the time course. The
proceeded, were at picomolar order or lower. Accordingly, time course experiments were repeated several times. All
13
the mechanism of packaging DNA described here does not the pfu measurements were consistent. The plate with the
rely on ATP, giving rise to an ATP-free packaging system. most complete set of pfu values was selected. Inoculated
Virions that are produced by this system are the actively plates were incubated at 36°C for 12 h before counting. The
infectious, at a level equivalent to the infectivity of original changes in the pfu values of T4 samples were monitored
population. The conformational change of DNA molecules regularly to confirm the activity of the T4 virion during
caused by the relative balance of the divalent cations, Ca , the experiments.
2+
and Pi 14,15 is the potential mechanism underlying these
ejection-packaging processes. 2.4. Fluorescent light microscopic observation

2. Materials and methods Virion particles and ejected DNA were distinguished
with fluorescent microscopy. Aliquots of the suspensions
2.1. Strains of virions and DNA were collected on 0.02 μm Anodisc
The bacteriophage investigated in this study was T4 (ø25 mm), backed by a pre-moisturized filter (Millipore
(ATCC 11303-B4), and its host bacteria were Escherichia HA). Virions and DNA collected on the filters were stained
coli (ATCC 11303). with ca. 30 μL of 1000× diluted SYBR-Gold solution
(Molecular Probes, Inc.), which comprised 0.1 or 10 mM
2.2. Preparation of T4 virions phosphate buffer (pH 8.0), 0.1 mM EDTA (pH 8.0), and
Peptone broth (peptone 10 g; glucose 1 g; NaCl 3 g; 0.1 M 50 mM dithiothreitol (Wako Pure Chemical Ind. Ltd. for
CaCl 1 mL; 0.1 M MgCl 10 mL; 0.1 M KH PO 3.2 mL; molecular biology). Sample filters were mounted on a glass
2
2
4
2
in 1 L solution, pH 7.2) was used for culturing the host slide with a mounting medium composed of 50% glycerol
bacteria, E. coli. T4 suspension was obtained by plate lysate (Wako Pure Chemical Ind. Ltd., special grade), 0.1 or 10 mM
method and small-scale liquid culture. In the plate lysate phosphate buffer (pH 8.0) and 40mM dithiothreitol. The
16
method, the virions were extracted by adding 2 – 3 mL phosphate buffer concentration was selected in accordance
of an electrolyte solution (EL) containing 1.8 mM NaCl, with the viral specimen, that is, 10 mM for ejected DNA
0.12 mM MgSO , 0.12 mM MgCl , 0.034 mM CaCl , and and 0.1 mM for compact DNA. In addition, 10 mM MgCl
2
2
2
4
0.05 mM KCl and incubated for several hours to elute was added to the specimens of compact DNA. Samples were
virions. Finally, the eluent was filtered with 0.2 μm filter observed with Olympus BX50 epifluorescent microscope,
(Advantec AS020) to remove bacteria and bacterial debris. equipped with N.A. 1.35 UPlan Apo ×100 objective lens,
To exclude the effects of anonymous ions and ATP in the N.A. 0.4 UPlan Apo ×10 objective lens, and U-MWBV
packaging experiments, the suspensions of T4 virions dichroic mirror unit. Samples were prepared for at least
were purified with ultracentrifugation and dialysis. For two filters. At the beginning of enumeration, one sample
ultracentrifugation, crude bacteriophage particles were filter which showed even distribution of the specimens on
purified by isopycnic centrifugation through CsCl gradients the filter confirmed by a general view of the whole filter
(16; Beckman XPN-90, SW32 rotor, 4°C, 24 h). Following was chosen. Using this sample filter, enumerations were
ultracentrifugation, T4 suspensions were dialyzed with performed at more than 20 randomly selected fields along a
Nuclepore polycarbonate membrane filters (0.015 μm diameter from one point to another on the periphery. More
in pore size, Whatman Inc.) against 0.5 mM CaCl for than 200 objects were counted, except for the cases where
2
1 week, with the 0.5 mM CaCl solution replaced 5 times there were almost no visible objects.
2
Volume 1 Issue 1 (2024) 69 doi: 10.36922/mi.2666