Viral contribution to dissolved DNA in the marine environment as determined by differential centrifugation and kingdom probing.
Dissolved or filterable (<0.2-(mu)m-pore-size filter) DNA is a ubiquitous component of the dissolved organic matter in the surface waters of this planet. In an effort to understand the composition and possible sources, we subjected dissolved DNA concentrated by vortex flow filtration from offshore and coastal environments to differential centrifugation and probing with 16S rRNA-targeted kingdom oligonucleotide probes. Initial studies with calf thymus soluble DNA and T2 phage particles indicated that high-speed ultracentrifugation (201,000 x g for 90 min), a method to separate viral particles from soluble DNA used by other investigators, resulted in pelleting of nearly all the DNA and virus particles. Lower-speed centrifugation (11,200 to 25,800 x g for 90 min) resulted in >99% of the virus particles being collected in the pellet and (equiv)65% of the calf thymus DNA remaining in the supernatant. Employing this approach, we estimate that approximately 50% of the filterable DNA from marine environments is truly soluble or free DNA and that the other half is composed of bound forms (viral particles and, potentially, colloids). Of the bound form, 17 to 30% could be accounted for by viral particles, by calculating the amount of viral DNA on the basis of viral abundance, leaving a portion of the bound form uncharacterized. Kingdom probing with universal, eubacterial, and eucaryotic probes indicated that dissolved DNA hybridized with all of these probes, while purified standard viral DNAs did not, or hybridized only slightly with the universal probe (tailed oligonucleotide only). Collectively, these data indicate that DNA in viral particles is a small component of the dissolved DNA, the majority being of eubacterial and eucaryotic origin.
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Jiang
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Paul