Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates

Abstract

Diverse microbes release membrane-bound extracellular vesicles from their outer surfaces into the surrounding environment. Vesicles are found in numerous habitats including the oceans, where they likely have a variety of functional roles in microbial ecosystems. Extracellular vesicles are known to contain a range of biomolecules including DNA, but the frequency with which DNA is packaged in vesicles is unknown. Here, we examine the quantity and distribution of DNA associated with vesicles released from five different bacteria. The average quantity of double-stranded DNA and size distribution of DNA fragments released within vesicles varies among different taxa. Although some vesicles contain sufficient DNA to be visible following staining with the SYBR fluorescent DNA dyes typically used to enumerate viruses, this represents only a small proportion (<0.01-1%) of vesicles. Thus DNA is packaged heterogeneously within vesicle populations, and it appears that vesicles are likely to be a minor component of SYBR-visible particles in natural sea water compared with viruses. Consistent with this hypothesis, chloroform treatment of coastal and offshore seawater samples reveals that vesicles increase epifluorescence-based particle (viral) counts by less than an order of magnitude and their impact is variable in space and time.

Figure 1

Size distribution of DNA associated with extracellular vesicles from marine bacteria. Fragment size distribution of double-stranded DNA extracted from extracellular vesicles of cultured isolates of Salinicola (black), Alteromonas (orange) and Thalassospira (violet) compared with data for Prochlorococcus (green; Biller et al., 2014). Peaks for all strains at 35 bp and 10 kb are Bioanalyzer internal standards.

Figure 2

Quantitative analysis of vesicles visible by epifluorescence microscopy. SYBR Gold-visible particle concentrations were determined from cultures of the indicated bacteria and compared with the total vesicle concentration determined by nanoparticle tracking analysis. Values indicate mean±s.d. of three biological replicates.

Figure 3

Bounding the potential contribution of vesicles to estimates of viral abundance in marine samples based on SYBR staining. (a) Field sampling locations. (b) Impact of chloroform treatment on SYBR-based particle counts from three coastal sites. NS=not statistically significant (P>0.05, two-tailed t-test); the Bayboro Harbor (BBH) sample collected on 06 May 2014 exhibited a significant difference (P<0.05, two-tailed t-test). (c) Impact of chloroform treatment on samples from the Gulf of Mexico. All samples exhibited a statistically significant difference in particle abundance between untreated and chloroform-treated samples (P<0.05, two-tailed t-test). Data in (b) and (c) represent the mean±s.d. of two replicates.