A method for characterizing dissolved DNA and its application to the North Pacific Subtropical Gyre

Abstract

Dissolved DNA (D-DNA) is a ubiquitous component of dissolved organic matter in aquatic systems. It is operationally defined as the DNA that passes a membrane filter and thus includes pools of truly dissolved "free" DNA (F-DNA), virion encapsidated DNA, DNA within membrane vesicles, and possibly other bound forms, each with different sources and lability. We investigated whether filtration (< 0.1 μm), concentration by tangential flow ultrafiltration (> 30 kDa), and fractionation in an equilibrium buoyant density gradient could be used to discriminate the mass contributions of the different pools of filterable DNA in seawater. Spike-in experiments with a known range of DNA standards (75-20,000 bp) indicated that this method results in high recoveries of F-DNA (68-86%) with minimal degradation. Application of the fractionation method to seawater samples collected from the oligotrophic North Pacific Ocean followed by analysis of fractions (epifluorescence and electron microscopy, DNase digestion) suggested that the low-density fractions (1.30-1.35 g mL −1) were dominated by vesicle-like particles, mid-density fractions (1.45-1.55 g mL −1) by virus-like particles, and high-density fractions (1.60-1.70 g mL −1) by F-DNA. The estimated concentration of DNA that is either F-DNA, in viruses, or in vesicles was 0.13, 0.14, and 0.08 μg L −1 , respectively in the euphotic zone and 0.09, 0.04, and 0.03 μg L −1 , respectively in the mesopelagic zone. The approach described should be useful for more detailed investigations of the abundance, dynamics, and sources of DNA in the distinct pools that comprise filterable DNA in aquatic environments. Dissolved DNA (D-DNA)-a highly labile and nutrient-rich component of dissolved organic material (DOM)-is ubiquitous in all aquatic habitats investigated to date, including freshwater rivers and lakes (Pillai and Ganguly 1972), coastal marine and estuarine systems (DeFlaun et al. 1986), sediments (Dell'Anno et al. 2002), and the open ocean (Karl and Bailiff 1989; Brum 2005). Concentrations of D-DNA often represent a substantial fraction of the total DNA (DeFlaun et al. 1987; Karl and Bailiff 1989), especially in the open ocean. Spatial patterns of D-DNA concentrations in the ocean are similar to those of total microbial biomass and total DOM, decreasing with distance from shore and with depth (DeFlaun et al. 1987; Karl and Bailiff 1989; Weinbauer et al. 1993, 1995). The concentration and bioavailability of D-DNA has both evolutionary and ecological significance. Double stranded D-DNA has the potential to alter the genetic make-up of microbial cells by the process of natural transformation (Hermansson and Linberg 1994) or, if hydrolyzed, can serve as a source of nitrogen and phosphorus (Jørgensen and Jacobsen 1996), macronutrients that are often limiting to microorganisms in open ocean ecosystems (Karl and Church 2014). As a consequence, there has been considerable interest in developing methods to investigate the nature and dynamics of this component of the larger DOM pool. Methods to stud...

Microbial sources of exocellular DNA in the ocean

“…Averaged across all virus fraction metagenomic libraries, these samples were dominated by annotated sequences derived from viruses (Figure 1), consistent with previous reports of this D-DNA fraction utilizing transmission electron micrographs and epi uorescence analyses (29). The viral metagenomic libraries had the lowest number of recovered sequences, many of which were novel and unannotated (61-73% unannotated across all samples).…”

“…Exocellular microbial-derived DNA is not unique to the open ocean (3,7,29,40). Terminology may vary, but F-DNA has also been reported in marine sediments (41) and terrestrial soils (42), even contributing 90% of the total DNA pool in marine sediments (43).…”

“…Our research utilized a new method (29) for separating ecologically important pools of D-DNA, and preliminary analyses of DNA sequences found in water column pro les of the different fractions of vesicles, viruses, and exocellular F-DNA. The results highlight the potential ecological contributions of D-DNA with respect to the cycling of limiting nutrients (nitrogen and phosphorus) in the open-ocean, as well as carrying mobile genetic elements.…”

“…Primarily due to prior D-DNA isolation methods that utilized low-volume ltration (27) and chemical precipitation (7,28) to isolate bulk D-DNA, these methods pose challenges in acquiring enough DNA to assemble metagenomic libraries for sequencing, leaving at least 25% of the open ocean DNA inventory uncharacterized. The average proportion of D-DNA that is either F-DNA, in viruses, or in vesicles is, 45, 35, and 20%, respectively (29).…”

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

“…Trial and error are essential, and experimentation with the iodixanol range used in the gradient and ultracentrifugation conditions will be required to maximize the separation; a of smaller volume, more finely resolved density fractions may also be required. Depending on the context, using CsCl gradients instead of iodixanol may help separate environmental particles 45 . Still, the change in osmotic conditions could lead to biases in the final recovered products, as discussed above.…”