Microbial Sources of Exocellular DNA in the Ocean

Abstract

Exocellular DNA is operationally defined as the fraction of the total DNA pool that passes through a membrane filter (0.1 μm). It is composed of DNA-containing vesicles, viruses, and free DNA and is ubiquitous in all aquatic systems, although the sources, sinks, and ecological consequences are largely unknown. Using a method that provides separation of these three fractions, we compared open ocean depth profiles of DNA associated with each fraction. Pelagibacter-like DNA dominated the vesicle fractions for all samples examined over a depth range of 75 to 500 m. Viral DNA consisted predominantly of myovirus-like and podovirus-like DNA and contained the highest proportion of unannotated sequences. Euphotic zone free DNA (75 to 125 m) contained primarily bacterial and viral sequences, with bacteria dominating samples from the mesopelagic zone (500 to 1,000 m). A high proportion of mesopelagic zone free DNA sequences appeared to originate from surface waters, including a large amount of DNA contributed by high-light Prochlorococcus ecotypes. Throughout the water column, but especially in the mesopelagic zone, the composition of free DNA sequences was not always reflective of cooccurring microbial communities that inhabit the same sampling depth. These results reveal the composition of free DNA in different regions of the water column (euphotic and mesopelagic zones), with implications for dissolved organic matter cycling and export (by way of sinking particles and/or migratory zooplankton) as a delivery mechanism. IMPORTANCE With advances in metagenomic sequencing, the microbial composition of diverse environmental systems has been investigated, providing new perspectives on potential ecological dynamics and dimensions for experimental investigations. Here, we characterized exocellular free DNA via metagenomics, using a newly developed method that separates free DNA from cells, viruses, and vesicles, and facilitated the independent characterization of each fraction. The fate of this free DNA has both ecological consequences as a nutrient (N and P) source and potential evolutionary consequences as a source of genetic transformation. Here, we document different microbial sources of free DNA at the surface (0 to 200 m) versus depths of 250 to 1,000 m, suggesting that distinct free DNA production mechanisms may be present throughout the oligotrophic water column. Examining microbial processes through the lens of exocellular DNA provides insights into the production of labile dissolved organic matter (i.e., free DNA) at the surface (likely by viral lysis) and processes that influence the fate of sinking, surface-derived organic matter.

Keywords: bacteriophage; bacterioplankton; eDNA; exocellular DNA; free DNA; metagenomics; microbial ecology; microbial oceanography; open ocean; vesicle.

FIG 1

Taxonomic annotation of metagenomic sequences from the three exocellular DNA constituents collected from the North Pacific Subtropical Gyre. Pie charts along the top display the domain-level taxonomic composition and proportion of unannotated sequences for each sample. Stacked bar charts represent the family-level taxonomic compositions of the three isolated exocellular DNA constituents (vesicles, viruses, and free DNA). The vesicle constituents were collected from three euphotic and mesopelagic zone depths (75, 125, and 500 m) in the North Pacific Subtropical Gyre. Viral fractions were collected from five depths (75, 100, 125, 250, and 500 m). Free DNA samples were collected from six depths (75, 100, 125, 250, 500, and 1,000 m).

FIG 2

Annotation of exocellular DNA metagenomic sequences suggests their probable depths of origin. The best sequence matches to the ALOHA 2.0 gene catalog (and their corresponding sampling depths) were used to assign the probable depth of origin to individual exocellular DNA metagenomic sequence reads. The size of the pie chart is proportional to the total number of exocellular DNA metagenomic reads, whose best match is to a Station ALOHA gene originating from a given corresponding water depth. Eukaryotes are excluded due to a lack of visibility, and all pie chart proportions are <0.004 for all samples and depths.

FIG 3

Fragment analysis of free DNA samples from the North Pacific Subtropical Gyre. The relative fluorescence units of each sample were used to normalize and calculate proportions. Molecular weight values are shown from 75 to 50,000 bp according to the Agilent DNF-488-0500 protocol sizing range. Samples collected above the deep chlorophyll maximum (125 m) had more distinct peaks and less DNA outside the peaks, while samples below had a broader distribution of sizes, indicative of degradation.