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Microbes & Immunity An ATP-free packaging of T4 DNA

abundant coil and globular DNA molecules were observed but not the coil DNA in 30 mM Pi, the intra-cell mimic
after the DNase I treatments and no decrease in pfu counts concentration of Pi. After 2 days of the above preparations,
was observed (Figures 1A and 4E). Consequently, we the on-filter-dry preparation of T4 virions in EL decreased
compared the DNase I degradation in situ and on-filter in size dramatically, while the on-filter-wet preparations of
versus the suspensions of T4 virions in EL, 30 mM Pi, and T4 virions in EL and coil or globular DNA molecules in
TE (Figure 4). As described in the Materials and methods 30 mM Pi did not change the FLM condition (Figure 6).
section, in in situ treatment, the DNA molecules in the The compact DNA molecules in the capsids were gradually
original solvents were degraded, that is, in EL, 30 mM Pi digested by DNase I after the virions were exposed to air.
and TE, and in on-filter-dry treatment, the suspensions Probably, the virion heads were deformed by the exposure
were filtered once and DNA molecules collected on 0.02 to air and a trace of DNase I might have seeped into the
μm Anodisc to remove the original solvents. Through heads to digest packaged DNA molecules. Modification
this filtration, coil DNA molecules and virion particles of DNA may protect T4 DNA from nuclease digestion.
34
were exposed to air and adsorbed on the filter surface. The protective effect on T4 DNA against DNase I is
Afterward, the collected DNA molecules and virion possible when the DNA is in coil form in intracell mimic
particles were degraded on-filter with DNase I in EL or concentrations of Pi, but not when it is attached on a filter
10 – 30 mM Pi, according to the original suspensions. In or in the compact conformation. The inactivity of DNase I
on-filter-wet treatment, the suspensions with DNase I were in 30 mM Pi also can be explained by the lack of enough
mounted on 0.02 μm Anodisc and filtered slowly. When the concentration of metal ions, for example, Mg , Ca , and
2+
2+
sample suspension decreased, additional DNase solution Mn , which are necessary for DNase activity, 35,36 due to the
2+
was supplied. Through this filtration method, coil DNA chelation of metal ions by Pi. While DNase I in 10 – 30 mM
molecules and virion particles were filtered on the filter Pi digests coil DNA attached on a filter, the concentrations
surface but not exposed to air during DNase degradation. of metal ions in 10 – 30 mM Pi are high enough for the
In the T4 suspensions in EL, DNase I treatment did not activity of DNase I. The behaviors of ejected DNA in
change the FLM abundances and the pfu counts among the 30 mM Pi and in TE were also somewhat different. The
original specimens, in situ and on-filter-dry DNase I-treated coil DNA molecules in 30 mM Pi showed active Brownian
specimens (Figure 4A-C). On-filter-wet DNase I treatment motion during microscopic observation, while the ejected
also did not change the FLM abundance (data not shown). DNA molecules in TE were more likely to stick on the
Intact virions were not digested with DNase I. The ejected surface of the filter.
DNA in 30 mM Pi was not digested through in situ DNase
I treatment (Figure 4E). During the DNase I treatment, The high pfu abundances in DNase I-treated 30 mM
parts of DNA molecules became globular (Figure 4E), Pi suspensions are not derived from the intact virions in
30 mM Pi suspensions, which are resistant to DNase I,
becoming bigger than intact virions (Figure 4A-C), and it is but originated from DNA-ejected virions in 30 mM Pi,
known that the globular conformation is more stable than which convert into infective virions during the plating
coil conformation and DNA in the latter conformation
15
can naturally transform into the former conformation. processes that dilute the concentration of phosphate and
On-filter-dry DNase I in 10 mM and 30 mM Pi treatments re-produce infective virions. The ejected DNA molecules
from virions in situ are resistant to degradation by DNase I
digested almost all DNA molecules (Figure 4F), while (Figure 4D and E) and maintain their intact physiological
on-filter-wet DNase I in 10 mM and 30 mM Pi treatments
did not digest coil or globular DNA molecules (data not activity (Figure 5). The re-production of infective virions
shown). This indicates that: (i) the DNase I in 10 mM and during the plating of DNA-ejected T4 suspension on the
30 mM Pi maintained the ability to degrade DNA, and peptone agar plate not only recovers infectivity but also
converts some non-infective virions into infective virions.
(ii) almost no intact virion survived in 30 mM Pi. The
abundances of DNase I resistant DNA particles in 30 mM The prevailing accepted theory is that DNA packaging
Pi, determined by FLM, were ca. 1% or less of the original of most double-stranded DNA bacteriophages commences
viral abundances (Figures 1 and 4). In contrast, few when a terminase creates an end of the concatemeric DNA,
globules of DNA remained after DNase I treatment in TE which attaches to the portal vertex of a capsid and packages
(Figure 4H), while both the initial population in TE- and the self-repulsive DNA string into the empty capsid with a
DNase I-treated suspensions showed no infectious ability. molecular motor, fueled by the energy produced from ATP
All the DNA of T4 in TE was degraded with the on-filter- hydrolysis. 3,32,37,38 This type of ejection-packaging system
dry DNase I treatment (Figure 4I). It becomes clear that is hereinafter referred to as the “motor-ATP system.”
DNase I, an endonuclease from bovine pancreas, digests Accordingly, the motor-ATP system cannot work without
single- and double-stranded DNA, and T4 DNA in TE, ATP. 5

Volume 1 Issue 1 (2024) 76 doi: 10.36922/mi.2666