DNA containing extracellular vesicles leading to confusion

The possibilities of cells to communicate and interact with each other and their surrounding are vast. Biofilms are a great example – communication appears on different levels. Cells interchange biomolecules via direct physical contact, release chemical molecules or regulate the expression of genes as a response to the surrounding cell density, known as quorum sensing (Miller and Bassler, 2001). Another way to communicate for microorganisms is the excretion of extracellular vesicles (EVs). Cells of all domains of life produce EVs. These tiny structures, defined by a lipid-membrane can contain lipids, proteins, nucleic acids such as DNA fragments or other biomolecules. The significance of these small “bubbles” regarding biological processes is impressive. Apart from enabling intercellular communication, EVs play a role in pathogenesis, the acquisition of nutrients, in biofilms or can even be part in the cellular defence mechanisms.

Due to the appearance and other characteristics EVs are hard to distinguish from viruses. When those biological particles were first discovered concerns arose in the world of viral research regarding a widely used method for the measurement of viral abundance in seawater. The basis of this standard approach is a fluorescent DNA-binding dye, often SYBR dye. DNA-containing EVs could have been detected via this method unknowingly leading to overestimated numbers of viral abundance.

In the presented study, the main aim was to find out more about DNA containing extracellular vesicles by measuring the size and frequency of fragments and to evaluate the standard method of DNA-binding epifluorescence for the assessment of viral abundance.

The organisms of interest were four different gram-negative heterotrophic bacteria species (Prochloroccocus, Salinicola, Alteromonas and Thalassospira), cultivated as axenic cultures.

Interestingly the DNA content varied significantly among the four marine bacteria. Each of the released DNA fragments possessed unique and species-specific size distribution, between 35bp and 10kb. With the use of SYBR-staining the researches observed a heterogeneous DNA distribution, meaning the fragments were not uniformly scattered among individual vesicles. Therefore only <0.1 vesicles were identified with that method. Exclusively vesicles with large DNA-fragments could be visualized.

The investigated differences in size and amount of DNA fragments on a species-level indicate that vesicles may have a high potential to act as agent of horizontal gene transfer. The researchers underline the importance to look deeper into this subject, because DNA-containing EVs may have a significant effect in the marine environment by acting as defence agents or “vehicles” of transport between targeted host cells of viruses leading to a change of sensitivity to those pathogens.

To assess the questioned method for viral counting and the applicability in the field, seawater samples were collected. Half of the samples were treated with chloroform, which disrupted the lipid-membrane structure of EVs, to enable a first separation of vesicles and viruses. The other half was kept as a control. Eventually the chloroform did not have a significant impact on counts of particles. In sum, the epifluorescence method only led to a fraction of visualized vesicles in the laboratory. Combined with the field-based work where a relatively small decrease in SYBR-bound particles appeared upon chloroform treatment, EVs do not impact the estimates of viral abundance in marine waters notably.

Conclusively, this study is a great example demonstrating that a high quality data collection and analysis needs suitable methods. With new findings previous methods should be questioned, examined, evaluated for they furthermore applicability. Regarding EVs more research is needed in order to understand the exact mechanisms of fragment and vesicle production and to work out the role of EVs on an ecological level to a greater extend.

Article reviewed:

Biller, S. J., McDaniel, L. D., Breitbart, M., Rogers, E., Paul, J. H., & Chisholm, S. W. (2017). Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates. The ISME journal, 11(2), 394.

References:
 

Miller, M. B., & Bassler, B. L. (2001). Quorum sensing in bacteria. Annual Reviews in Microbiology, 55(1), 165-199.

Schatz, D., & Vardi, A. (2018). Extracellular vesicles—new players in cell–cell communication in aquatic environments. Current opinion in microbiology, 43, 148-154.