Use of PEG, Polyethylene glycol, to characterize the diversity of environmental viruses

Jonathan Colombet and Télesphore Sime-Ngando1

Laboratoire Microorganismes : Génome et Environnement, Clermont Université Blaise Pascal, UMR CNRS 6023, 24 avenue des Landais, BP 80026, F-63171, Aubière Cedex, France 1Corresponding author: Phone: + 33 4 73 40 78 36 ; Fax: + 33 4 73 40 76 70. E-mail: Telesphore.SIME-NGANDO@univ-

bpclermont.fr

This article focuses on the use of polyethylene glycol (PEG) as a virioplankton concentrating agent to characterize the diversity of natural communities of aquatic viruses. PEG is well known as good inductor of attractive interactions that crystallize viruses in the interpolymer spaces between PEG molecules. Added to a co-precipitant it allows an easy and rapid concentration of virioplankton. A final step of purification to remove PEG allows obtaining virioplankton concentrates of optimal quality (i) to characterize the genomic diversity of viruses via Pulsed Field Gel Electrophoresis or other molecular analyses, and (ii) to observe viral morphotypes using transmission electronic microscopy (TEM). Experimental procedure described was successfully applied on various fresh- or salt-water ecosystems exhibiting different physicochemical and biological conditions.

Keywords: Virioplankton; Concentration/purification; Polyethylene glycol (PEG); Transmission electronic microscopy (TEM); Molecular techniques

1. Introduction

Phages are now recognized as ubiquitous, abundant, and diverse biological entities in world aquatic ecosystems where their activity is crucial to several processes, including microbial mortality, potential gene transfer, and lysogenic conversion [see 1-2 for review]. Metagenomic analysis of viral communities collected in four major oceanic regions over a decade showed a high global diversity, presumably several hundred thousands of ‘species’ [3]. However, the genetic and biological diversity of aquatic viruses remains largely unexplored, mainly because of the methodological difficulties related to the observation of environmental nanoparticles and the weakness of environmental viral gene bank. The study of free-floating viruses in aquatic systems requires a concentration method for estimating their number and their diversity, i.e. based on both morphologic and genomic features. The first generation of methods used came from the study of human, animal and plant pathogenic viruses. These methods are based on physicochemical approaches such as adsorption of phages onto microporous filters charged positively or negatively [4-5], the use of precipitation agents like calcium phosphate, aluminium or magnesium or acid precipitation [6], and the use of organic flocculation [7]. The main disadvantage of these techniques for natural samples is their selectivity. Indeed, various viruses may have different adsorptive properties [8], and electrostatic interactions may affect the viability of concentrated viruses. Chemical precipitation methods generally require low pH that may also compromise the viability of some viruses [9]. Børsheim et al. [10] have proposed a concentration method based on the ultracentrifugation of viruses contained in few milliliters of samples directly onto grids, followed by staining before observation under transmission electron microscope (TEM). Disadvantages of this method include the risk of viral disruption due to the high centrifugation speed, the effects of suspended particles and ‘virucidal’ substances, and the low initial volume of experimental samples that may render TEM observations tedious according to the high magnifications used. The method is thus time-consuming (for large volume of samples) and costly, and therefore cannot be recommended as a routine procedure [11]. An alternative approach is the one currently used for aquatic microbes, based on low pressure membrane-concentration, fluorochrome staining, and the use of epifluorecence microscope [11-12]. A protocol using fluorochrome staining and flow cytometer [13] was optimized for rapid counting of virus-like-particles (VLPs) in the plankton [14]. However, these fluorochrome-based methods are well known to provide accurate counts but poor description of microbial viral diversity. An accurate description of the diversity of pelagic viruses usually needs highly concentrated aliquots. The current method available to date is ultrafiltration that allows concentrating viruses contained in several tens or hundreds of liters in small volume of some tens of milliliters [15-16]. However, the application of this procedure must be followed by ultracentrifugation to obtained viral pellets necessary for TEM observations and for estimating viral diversity [17-19]. To avoid ultracentrifugation and the underlined disadvantages, Colombet et al. [20] proposed a simple, cheaper and efficient alternative protocol using polyethylene glycol (PEG) concentration to obtain virioplankton concentrates that can be used for different purposes, primarily for TEM observations, electrophoretic plugs, and the following cloning/sequencing possibilities for molecular analyses. Compared to ultracentrifugation this protocol allowed, on average, the recovery of 2-fold more viruses. In this paper we briefly describe the procedure of

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concentration/purification of virioplankton using PEG, in the general context of virioplankton diversity studies in fresh- and salt-water ecosystems. PEG and monovalent salts are well known as good inductors of attractive interactions that preferentially crystallize biological macromolecules, such as DNA and viruses, in the interpolymer spaces between PEG molecules (see [21] for the logic behind). PEG was first used by Albertson and Frick [22] and Philipson et al. [23] to precipitate viruses between two immiscible aqueous polymer phases. Since then, PEG has been used, usually in combination with salts such as NaCl, to recover various viruses in different growing medium [9].

2. Origin of experimental samples

Virioplankton concentration protocol using PEG described in experimental procedure was used for viral genomic and/or morphological viral diversity characterization in various environments covering a wide range of physicochemical and biological conditions, including pelagic samples from the oligomesotrophic Lake Pavin (France), the mesotrophic Lake Bourget (France) and the shallow hypersaline Lake Retba (Senegal), and the deep anoxic sediments of Lake Pavin. The main characteristics of these study sites are provided in Table 1.

Table 1. Characteristics of the environments under study.

Lake Bourget (France)

Lake Pavin (France)

Lake Pavin (France)

Lake Retba (Senegal)

Sampling area

Oxygenated pelagic freshwater(a)

Oxygenated pelagic freswater(b)

Freshwater anoxic sediment(c)

Hypersaline pelagic water(d)

Sampling date

July 2008 April to December 2005; June 2008

December 2009 April 2008

Coords 45°43’47”N, 5°52’10”E

45°29’41” N, 2°53’13”E

45°29’41” N, 2°53’13”E

14°50′14″N, 17°14′55″W

Trophic status

Mesotrophic Oligomesotrophic Oligomesotrophic

Depth 145 m 92 m 92 m 1.5 m

Area 44.5 km² 0.44 km² 0.44 km² 3 km²

Altitude 231.5 m 1197 m 1197 m

Drainage basin

56 000 ha 50 ha 50 ha

Bacterial abundance

3.106cells ml-1(a) 6.106cells ml-1(b) 5.109 cells g-1 dry sediment (c)

3.108cells ml-1(d)

Viral abundance

3.107particles ml-1(a) 2.107particles ml-1(b) 21.109particles g-1 dry sediment (c)

7.108particles ml-1(d)

(a), reference 33 - Sime-Ngando T, Colombet J, Domaizon I, Personnic S, Dorigo U, Perney P, Viollier E, and Jacquet S (2008) Short-term variations in abundances and potential activities of viruses, bacteria and nanoprotists in lake Bourget. Ecological research, 23: 851–861 (b), reference 20 - Colombet J, Charpin M, Robin A, Portelli C, Amblard C, Cauchie HM, Sime-Ngando T (2009) Seasonal Depth-Related Gradients in Virioplankton: Standing Stock and Relationships with Microbial Communities in Lake Pavin (France). Microbial Ecology, 58:728-736. (c), reference 25 - Borrel G, Colombet J, Robin A, Lehours AC, Prangishvili D and Sime-Ngando T (2012) Unexpected and novel putative viruses in the sediments of a deep-dark permanently anoxic freshwater habitat.The ISME Journal, doi:10.1038/ismej.2012.49. (d), reference 28 - Sime-Ngando T, Lucas S, Robin A, Tucker KP, Colombet J, Bettarel Y, Desmond E, Gribaldo S, Forterre P, Breitbart M and Prangishvili D (2010) Diversity of virus–host systems in hypersaline Lake Retba, Senegal. Environmental Microbiology,13: 1956–1972.

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3. Experimental procedure

3.1 Viral concentration

All sampled were submitted to successive prefiltration steps (0.2-µmpore-size filter at the final stage). Environmental samples with viral concentrations below 108 viruses per millilitre (pelagic area from Lake Pavin and Bourget) were initially concentrated by ultrafiltration. Viruses contained in 20 L experimental samples were concentrated to a volume of approximately 1 L, using a high performance concentration/diafiltration system (Model DC 10LA, Amicon®) equipped with a reusable hollow fiber cartridge of 30-kDa cut-off and surface area of 0.45 m2 (Amicon®, Epernon, France). Recirculation rate was at 10 L min-1 with an entry pressure of 0.9 bar. For environmental samples naturally charged in virus (concentrations above 108 viruses per milliliter), namely the hypersaline lake Retba and the sediments of Lake Pavin, concentration by ultrafiltration was not necessary. Viruses in the sediments were extracted as described in detail in Borrel et al. [24]. For both concentrated (i.e. ultrafiltration retentates) and raw samples, Polyethylene glycol 8000 (PEG, catalog no. 81268; Sigma) together with NaCl (not added in hypersaline water) were added to 210 mL to final concentrations of 10 and 0.6%, respectively, and incubated at 4 °C in the dark, in a transparent cylindrical glass container of 400 mL, for at least 20 h. PEG molecules of molecular weight (mw) lower than 6000 are less effective in precipitating macromolecules, while molecules equaling or above 6000 mw appear to behave identically [25]. At the end of incubations, the white phase containing crystallized viruses (approximately 40 mL) was pipetted, transferred into a 50 mL sterile tube (BD Falcon™, Le Pont de Claix, France), centrifuged at 8000×g for 20 min at 4 °C, and resuspended in 300 μL of SM buffer [0.1 M NaCl, 8 mM MgSO4–7H2O, 50 mMTris–HCl, and 0.005% (wt/vol) glycerol) adjusted at neutral pH]. Viral solution was transferred into a 2 mL sterile tube (Eppendorf®, Le Pecq, France). 1 M KCl was then added, the mixture incubated on ice for 20 min and centrifuged (12,000×g, 10 min at 4 °C), in order to precipitate the PEG solution slowly while leaving the purified free viruses behind. The supernatant with clean viruses was finally pipetted, yielding a final volume of about 400 μL viral concentrate.

3.2 Analysis of viral diversity

Viral diversity in concentrates were estimated following different approaches: (i) pulsed field gel electrophoresis (PFGE) in Lake Pavin [26], (ii) Random Amplification of Polymorphic DNA (RAPD) in the sediments of Lake Pavin [24], (iii) metagenomics in Lakes Bourget and Pavin [27] and (iv) transmission electronic microscopy (TEM) in Lakes Pavin and Retba [24, 26, 28]. Genomic analysis of viral diversity by PFGE were performed directly on the viral concentrate after boiling in a waterbath at 60 °C for 10 min for nucleic acid extraction. Analysis by RAPD or metagenomics needed re-filtration of viral concentrates onto 0.2 µm followed by DNAse I (Invitrogen) treatment and DNA purification using Qiamp DNA mini kit (Qiagen). RAPD was performed after Polymerase Chain reaction (PCR) whereas viral metagenome was obtained after nonspecific amplification and single pyrosequencing. Possible bacterial contamination was checked by PCR amplification using the 16S rDNA primers 27f and 1492r as previously described [29]. For morphological analyses of viruses 60 µL of viral concentrates (stored at 4 °C) was diluted in 6 mL of 0.02 µm filtered distilled deionised water, and centrifuged at 120,000 x g for 2 h, 4 °C with a SW 40 Ti rotor (LE 80K, Beckman) onto 400-mesh Cu electron microscopy grid with carbon–coated Formvar film (Pelanne Instruments, Toulouse, France). Each grid was stained at room temperature for 30 sec with uranyl acetate (2% w/w), rinsed twice with 0.02 µm distilled water and dried on a filter paper. Observations were performed on a JEM 1200EX TEM (JEOL) operated at 80 KV at a magnification of 15 000 to 200 000 x. Post concentration treatments and the complete procedures for viral diversity analyses are detailed in Colombet and Sime-Ngando [26], Roux et al. [27], Borrel et al. [24] and Sime-Ngando et al. [28] for Lake Pavin, Lake Bourget, sediments of Lake Pavin, and Lake Retba, respectively.

4. Results and Discussion

Results obtained from the four considered environments are described and discussed in Colombet and Sime-Ngando [26], Roux et al. [27], Borrel et al. [24] and Sime-Ngando et al. [28] for Lakes Pavin, Bourget, the sediments of Lake Pavin, and Lake Retba, respectively. In this paper we focus on the application of virioplankton concentration/purification procedure using PEG in various environments and for different study purposes.

4.1 Viral genomic diversity

Analyses of viral genomic diversity by PFGE, RAPD or metagenomic analyses in the study sites clearly showed that the PEG procedure allowed to obtain viral DNA suitable for determination of viral genomic size classes (PFGE) or for PCR amplification (RAPD, metagenome). The quantity of DNA obtained by this procedure was largely enough (> 5 x 109 viruses per ml) to perform analyses of viral genomic diversity by PFGE (see example of fingerprint in Figure 1). The

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quality of profile obtains and the absence of bacterial contamination showed that experimental procedure was efficient to obtain a high quality viral DNA concentrate suitable for DNA amplification (see example of RAPD fingerprint in Figure 2).

Figure 1. Virioplankton PFGE fingerprints for samples obtained from April to December 2005 in Lake Pavin. Months are indicated in the bottom of the gels and genome size on the left side of the gels. Mid = MidRange PFG Markers, Ap = April, Ma = May, Jun = June, Jul = July, Au = August, Se = September, Oc = October, No = November, De = December. More details are provided in Colombet and Sime-Ngando [26].

Figure 2. Virioplankton RAPD fingerprints for samples obtained from anoxic sediments of Lake Pavin in December 2009. Analyzed sediment depths (in cm) are indicated on the left side of the gel. More details are provided in Borrel et al. [24].

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4.2 Viral morphotypic diversity

Numerous observations of viral concentrates obtained after viral concentration/purification by PEG procedure showed that this rapid and inexpensive methodology allowed an easy characterization of virioplankton morphological diversity. Here we showed that this procedure was applicable on virioplanktonic communities from various environments, exhibiting different physicochemical and biological characteristics (Table 1): hypersaline (Fig.3 A) or freshwater lakes (Fig.3 B, Fig.4 a to l) and anoxic sediments (Fig.4 m to w). Atha and Ingham [30] reported that PEG precipitation was effective at various conditions, e.g. neutral pH, high ionic concentrations, and in the absence of other organic materials. Procedure described in this paper was successfully used by Bettarel and coauthors [31] to determined viral morphology diversity along a full-salinity gradient.

Figure 3. Overviews of virus-like particles observed by TEM in the environmental samples from the hypersaline Lake Retba (April 2008) (A) and the mesotrophic Lake Bourget (July 2008) (B) after PEG concentration/purification procedure. Samples were negatively stained with 2% uranyl acetate. More details are provided in Sime-Ngando et al.[28] and Roux et al.[27] for Lake Retba and Lake Bourget, respectively. Obtained photographs were of excellent contrast (Fig. 3 and 4) and details of viral structure were clearly identifiable (see example in Fig. 4). Moreover the low mechanic shocks permitted the preservation of the tails of phages (for example Fig. 4a-l, q-t). Finally this methodology was not selective since we identified a large range of different viral morphotypes in the virioplankton concentrates such polyhedric (Fig.3A and B, Fig.4 a to t), spindle (Fig.3A, Fig.4 u to w), and linear (Fig.3A) shaped viruses. All the range of known viral size was also recovered (Fig.3A and B, [26, 31]). Fine descriptions of viral morphotypes, including unexpected and novel types of viruses for freshwaters, are detailed in Borrel et al. [24], Colombet and Sime-Ngando [26], and Sime-Ngando et al. [28]. Rapid, inexpensive and non-destructive properties of the PEG procedure added to its non selectivity were previously reported for various specific viruses [9, 23]. It is important to note that the purification step using KCL is essential to remove PEG. Indeed PEG alone yields dirty, poorly contrasted, even opaque images with little structural details. Ackermann and Tiekotter [32] reported that when using PEG alone for a specific virus, four washing steps were needed to obtain a clean preparation.

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Figure 4. Overviews of planktonic virus observed by TEM in the environmental samples from pelagic area (a to l) (April to December 2005) and sediments (m to w) (December 2009) of the oligomesotrophic Lake Pavin after PEG concentration/purification procedure. Samples were negatively stained with 2% uranyl acetate. More details are provided in Borrel et al. [24].

5. Conclusions

In this paper we show that concentration/purification procedure of virioplankton using PEG is easy, rapid, inexpensive and suitable for about the whole physicochemical and biological condition of environmental samples. The quality of obtained virioplankton concentrates allows accurate characterization of virioplankton diversity by molecular and microscopic analyses. The PEG treatment, i.e. the so called pegylation, thus offers a valid, simple and cheaper alternative method to ultracentrifugation for the concentration and purification of environmental viruses.

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