Isolation and Staining Reveal the Presence of Extracellular DNA in Marine Gel Particles

Abstract

Marine gel particles (MGP) are amorphous hydrogel exudates from bacteria and microalgae that are ubiquitous in the oceans, but their biochemical composition and function are poorly understood. While dynamic ecological interactions between marine microorganisms and MGPs may result in the secretion and mixing of bacterial extracellular polymeric substances (EPS) such as nucleic acids, compositional studies currently are limited to the identification of acidic polysaccharides and proteins in transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP). Previous studies targeted MGPs isolated by filtration. We developed a new way of isolating MGPs from seawater in liquid suspension and applied it to identify extracellular DNA (eDNA) in North Sea surface seawater. Seawater was filtered onto polycarbonate (PC) filters with gentle vacuum filtration, and then the filtered particles were gently resuspended in a smaller volume of sterile seawater. The resulting MGPs ranged in size from 0.4 to 100 µm in diameter. eDNA was detected by fluorescent microscopy using YOYO-1 (for eDNA), with Nile red (targeting cell membranes) as a counterstain. TOTO-3 was also used to stain eDNA, with ConA to localise glycoproteins and SYTO-9 for the live/dead staining of cells. Confocal laser scanning microscopy (CLSM) revealed the presence of proteins and polysaccharides. We found eDNA to be universally associated with MGPs. To further elucidate the role of eDNA, we established a model experimental MGP system using bacterial EPS from Pseudoalteromonas atlantica that also contained eDNA. Our results clearly demonstrate the occurrence of eDNA in MGPs, and should aid furthering our understanding of the micro-scale dynamics and fate of MGPs that underly the large-scale processes of carbon cycling and sedimentation in the ocean.

Keywords: Coomassie stained particles; Pseudoalteromonas atlantica; bioimaging; extracellular DNA; extracellular polymeric substances; marine gel particles; staining; transparent exopolymeric particles.

Figure 1

Marine gel particles recovered from PC filters after re-suspension in 0.2 µm filter sterilised seawater. Scale bar = 20 µm, image captured at 63× objective and visualised with phase contrast (A). Scale bar = 50 µm (B–D), image captured at 40× objective for (B) and 20× for (C,D) and visualised using bright-field microscopy.

Figure 2

MGP from off the Northumberland coast (North Sea), visualised using bright-field microscopy with fibre light. The MGPs were stained with Alcian blue dye ((A,B); yellow arrows) and with Coomassie blue ((C,D); yellow arrows). Scale bar = 40 µm. Images captured using upright Leica microscope and fibre light with 10× objective.

Figure 3

Natural MGPs of different sizes from the North Sea. Bright-field microscopy images of MGPs (A) Nucleic acid SYTO 9 green stain of MGP revealing bacterial presence. Fluorescent staining with DNA dye SYTO 9 (1 nM) (B). Scale bar = 100 µm. Image capture using 20× objective.

Figure 4

CLSM images acquired using Leica SP8 of natural MGPs stained with YOYO-1 to localise the presence of eDNA (green). Nile red (red) was used as a counter stain for bacterial cell membranes. Scale bar = 5 µm (A). Image captured using 63× objective. Quantification of the fluorescence intensity of each fluorophore against the control (unstained MGPs) (B). The graph presents the mean ± SD of 15 CLSM micrographs. Error bars represent the standard deviation of the data set (n = 3), p < 0.05.

Figure 5

A 3D image of a single natural MGP stained with YOYO-1 (green = extracellular DNA) and Nile red (red = cell membrane). Scale bar = 5 µm. Image captured with Leica SP8 CLSM.

Figure 6

CLSM images of natural MGPs stained with TOTO-3 (red) to localise the presence of eDNA, SYTO 9 (green) for intracellular DNA and ConA (blue) for glycoproteins. Scale bar = 5 µm (A). Images acquired with Leica SP8 using 63× objective. Quantification of the fluorescence intensity of each fluorophore against the control (unstained MGP) revealed high mean value of TOTO-3 indicating the presence of eDNA (B). The control is unstained particles. The graphs present the mean ± SD of 15 CLSM micrographs. Error bars represent the standard deviation of the data set (n = 3), p < 0.05.

Figure 7

A 3D image of a single natural MGP stained with ConA (blue), TOTO-3 (red)-stained eDNA and SYTO 9 (green)-stained iDNA. Scale bar = 10 µm. Image captured with Leica SP8 CLSM.

Figure 8

Pseudoalteromonas atlantica biopolymers as MGP representative grown in artificial seawater after 96 h (A) and after 168 h (B). P. atlantica particles captured with inverted microscope using 20× objective. Scale bar = 330 µm (C).

Figure 9

P. atlantica EPS stained with Alcian blue ((A,B); yellow arrows) and Coomassie blue ((C,D); yellow arrows). Images taken using an upright Leica microscope. Scale bar = 50 µm.

Figure 10

CLSM images acquired using Leica SP8 at 63× of P. atlantica EPS MGP model stained for eDNA with YOYO-1 (green); Nile red (red) as counter stain for bacterial cell walls to localise the presence of eDNA. Scale bar = 5 µm (A). Quantification of the fluorescence intensity of each fluorophore revealed high mean value of YOYO-1, indicating the presence of eDNA. The control is unstained particles. The graphs present the mean ± SD of 15 CLSM micrographs. Error bars represent the standard deviation of the data set (n = 3), p < 0.05 (B).

Figure 11

A 3D image of P. atlantica particles stained with YOYO-1 (green) for eDNA and Nile red (red). Scale bar = 5 µm.

Figure 12

CLSM images acquired using Leica SP8 of P. atlantica EPS as MGP model stained for eDNA with TOTO-3 (red) and SYTO9 as counterstain (green), and ConA for glycoproteins (blue). Scale bar = 5 μm (A). Quantification of the fluorescence intensity of each fluorophore revealed a high mean value. The control is unstained particles. The graphs present the mean ± SD of 15 CLSM micrographs. Error bars represent the standard deviation of the data set (n = 3), p < 0.05 (B).

Figure 13

A 3D image of a single particle of P. atlantica particles stained with ConA (blue), TOTO-3 (red) and SYTO 9 (green). Scale bar = 10 µm.