Page 76 - MI-1-1

Microbes & Immunity An ATP-free packaging of T4 DNA

2.5. Ejection of T4 DNA 2.8. Digestion of capsid proteins by proteinase K

The ejection of DNA from T4 virion was conducted using To obtain capsids-free, naked DNA molecules of T4, the
biological (Pi, citrate, and an electrolyte mimicking cell capsid proteins of viral suspension were digested with
sap) and artificial (EDTA, Tris-HCl and TE [Nippon Gene proteinase K (Takara, ≥600 mAnson U/mL). Virions
20
Co., LTD]) chelates (pH = 8.0). An cell-sap-mimicking were suspended in EL, pH 8.0, with 1% v/v of proteinase
+
+
electrolyte is composed of 15mM Na , 140 mM K , K, which was incubated at room temperature for 1 day.
0.1 mM Mg , 10 mM Cl , 10 mM HCO , and 35 mM Tris, EDTA and SDS, which were normally included in the
-
2+
3-
HPO4 . The virions were exposed to combinations lysis buffer, were not composed in the present mixture of
2- 17,18
of counterion concentrations, that is, phosphate buffer proteinase K digestion solution proteinase K degradation
(pH 7.6) containing 0.01 – 100 mM Pi versus 0 – 1 mM to avoid any negative effects on pfu (discussed in section
CaCl or 0 – 1 mM Mg Cl , for several minutes. 3.2). Inactivated proteinase K, an enzyme solution heated
2 2
at 95°C for 15 min, was used as the control of the proteinase
2.6. Packaging of T4 DNA K treatment experiment.
The packaging of DNA into the capsids of T4 was induced
by 10 – 10 times dilution of the ejected specimens. The 2.9. Measurement of ATP concentration
5
solvent used for the dilution were 1 mM CaCl solution or The concentration of ATP was measured in triplicate.
2
EL. The diluted suspensions of T4 virions were incubated ATP in a specimen can be converted to adenosine
for ca. 10 min. The total number of reformed virions diphosphate (ADP) or adenosine monophosphate (AMP)
was measured visually with fluorescent light microscopy while processing the specimen; therefore, the measured
(FLM) and their infective ability was estimated by pfu concentration of ATP alone would be an underestimate.
counts using the plate method. The total concentration of ATP + ADP + AMP between
the ranges of 10 pM and 10 μM was measured by a reagent
2.7. DNase I treatment set, Lucipack A3 water, and Lumitester Smart (Kikkoman
DNase I (recombinant DNase I, Takara) treatment was Biochemifa Co.). Here, the ATP concentration was
applied to discriminate the DNA covered with capsid measured following the enzymatic conversion of ADP and
proteins from the naked DNA without capsid coverings. AMP to ATP, giving a value that is clearly an overestimate.
Two types of DNase I digestions were performed. One of Hereinafter, ATP concentration refers to the total amount
them was DNase I degradations in situ. DNase I was added of ATP, ADP, and AMP.
to suspensions of T4 in EL, 10 mM or 30 mM Pi and TE
with the concentrations of 1 U/40 μL plus 1 mM MnCl . 2.10. Statistical analysis
2
The specimens were incubated for 2 h. Statistical processing was conducted using Microsoft
The other treatment was on-filter degradation, which Excel 2016. Standard deviations of analyzed data were
can be conducted using two approaches, namely, on-filter determined. t-test was carried out with two-tailed
dry and on-filter-wet methods. During the on-filter-dry distribution and the two samples were assumed to have
treatment, suspensions of virions and DNA molecules equal variances.
were filtered on 0.02 μm Anodisc (ø25 mm), followed by 3. Results
mounting of DNase I solution, ca. 0.1 – 0.2 mL of 1 U/40 μL
19
dilution plus 1 mM MnCl in EL, and in EL plus 10 mM or 3.1. Ejection of DNA
2
30 mM Pi, on the filter to degrade naked DNA. Virions and The ejection of DNA from the head of a bacteriophage
DNA molecules were exposed to air once and adsorbed on was induced by incrementing the ambient concentration
the surface of the filter, and the original solvent was replaced of phosphate (Pi), a chelating agent. 8-10 When the
by DNase I solution in this process. During the on-filter- concentration of Pi and Ca was higher than 10 mM
2+
wet method, the in situ suspension was mounted on the and lower than 0.1 mM, respectively, the virions
filter, slowly filtered, and supplied with DNase solution instantly ejected their DNA (Table 1 and Figure 1). The
carefully when the suspension on the filter decreased. The intramolecular Brownian motion of the Gaussian random
DNA molecules and virions were filtered on the surface of coil DNA produced blurred ball images (Figure 1B).
a filter without being exposed to air. The DNase treatments Hydrodynamic flow of the mount medium stretched coil
were incubated for 0.5 – 1 h for on-filter-dry treatment and DNA and the stretched state was adhered to the filter
1 – 2 h for on-filter-wet treatment. surface by seeping mount medium including divalent
After incubation, specimens were filtered and stained cation from the backside (Figure 1B). The random
with SYBR-Gold. coil DNA was stretched to 10 – 20 μm long, which was

Volume 1 Issue 1 (2024) 70 doi: 10.36922/mi.2666